In Japan, two-step tuberculin skin test (two-step TST) is recommended for health care workers (HCWs) if the diameter of erythema in the first test is less than 30 mm, and to detect new infection if there is 10 mm or more increase from the base-line diameter. We observed TST in nurse students from the entrance to the graduation for 3 years, and analyzed the change of TST reaction to discuss the usefulness of two-step TST and the criteria for detecting new tuberculous infection among BCG vaccinated HCWs. Mean +/- S.D. (mm) in erythema and induration in each occasion were: in a group with single TST at entrance (T1) and before graduation (TG) (n = 99, group I); T1 = 19.8 +/- 11.3, TG = 23.6 +/- 14.3 for erythema and T1 = 12.5 +/- 5.3, TG = 13.8 +/- 5.0 for induration: in a group with two-step TST at entrance (T1 and T2) (n = 40, group II); T1 = 11.9 +/- 7.8, T2 = 20.4 +/- 10.6, TG = 22.1 +/- 13.5 for erythema and T1 = 8.6 +/- 6.1, T2 = 12.0 +/- 5.1, TG = 13.4 +/- 5.4 for induration: in a group BCG vaccinated after single negative TST (less than 10 mm of erythema and/or less than 5 mm of induration) (n = 12, group I-B); T1 = 4.0 +/- 3.5, TB = 19.8 +/- 5.8, TG = 16.3 +/- 7.6 for erythema and T1 = 0.5 +/- 1.5, TB = 14.1 +/- 3.7, TG = 11.9 +/- 4.4 for induration: in a group BCG vaccinated after two negative TST (n = 10, group II-B); T1 = 3.6 +/- 3.1, T2 = 6.2 +/- 2.4, TB = 23.9 +/- 8.5, TG = 14.1 +/- 6.9 for erythema and T1 = 1.7 +/- 2.7, T2 = 3.2 +/- 1.8, TB = 16.0 +/- 3.2, TG = 7.5 +/- 7.8 for induration. One student in the group II was diagnosed as tuberculosis before the graduation. If we exclude this case form the group II, mean +/- S.D. (mm) of TG in group II were 20.7 +/- 11.2 for erythema and 13.1 +/- 5.0 for induration. Booster phenomenon was significant on two-step TST. There was moderate booster phenomenon even after 34 months from the previous single TST. There was also significant waning of reaction 27 to 31 months after BCG vaccination. But there were no significant waning nor booster after two-step TST. Concerning the difference between the reaction before graduation and at entrance, mean +/- S.D. (mm) in erythema and induration, [TG-T1] in the group I were +3.8 +/- 11.4 and +1.6 +/- 5.8, respectively, [TG-T2 or 1] in the group II were +0.7 +/- 11.4 and +0.4 +/- 5.6, respectively, [TG-TB] in the group I-B and II-B were -6.4 +/- 9.9 and -5.0 +/- 6.5. If we exclude one case who got tuberculosis from the group II, mean +/- S. D. (mm) in erythema and induration of [TG-T2] in group II were -0.6 +/- 8.1 and +0.2 +/- 5.4, respectively. According to the criteria in Japan of more than 10 mm increase in erythema and more than 6 mm increase in induration, recommended by Menzies, significant values of [TG--(T1, T2 or TB)] was observed in 24 (24%) by erythema and 22 (22%) by induration in the group I, while in the group II only in 4 (10%) by erythema and 5 (12.5%) by induration, which included case diagnosed as active tuberculosis. The criterion of 10 mm increase in erythema seems to correspond to that of 6 mm increase in induration. 95% confidential limit in the differences between two tests were 15.6 mm (mean plus 2 standard deviation) in erythema and 11.0 mm in induration in the group II. Considering that these data may include some newly infected persons, appropriate criteria for detecting new tuberculosis infection is estimated to be between 10 to 15 mm increase in erythema and 6 to 11 mm increase in induration from the baseline by two-step TST. Among BCG vaccinated, TST two months after vaccination is useful as the base line. As there is moderate booster phenomenon even after three years from the previous single test and variation is more common, the detection of new tuberculous infection can be made more accurately with the two-step TST in HCWs.