Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of 6-mercaptopurine (6-MP) and other thiopurine drugs. The level of TPMT activity in human tissue is controlled by a common genetic polymorphism. Our experiments were performed to study the relationship between TPMT phenotype and the effect of 6-MP on [3H]thymidine ([3H]TdR) incorporation into mitogen-stimulated human peripheral blood lymphocytes. Lymphocytes were isolated from blood samples obtained from 12 subjects, four each with each of the three classes of TPMT phenotype. The effect of 6-MP concentration on [3H]TdR incorporation was determined in the presence of two mitogens, phytohemagglutinin at concentrations of 1 and 10 micrograms/ml and concanavalin A at concentrations of 1 and 5 micrograms/ml. ED50 values for 6-MP inhibition of [3H]TdR incorporation into lymphocytes from subjects who genetically lacked TPMT activity were uniformly higher than were ED50 values for lymphocytes from subjects with intermediate or high enzyme activities. However, in the presence of either mitogen, ED50 values decreased as the level of stimulation increased. Therefore, to make it possible to account for degree of mitogen stimulation, the effect of 6-MP on [3H]TdR incorporation was studied in the presence of a series of phytohemagglutinin concentrations from 1 to 10 micrograms/ml. Lymphocytes from subjects who genetically lacked TPMT activity had significantly higher Ki values (1.37 +/- 0.340 microM, mean +/- S.E.M., n = 3) for inhibition of [3H]TdR incorporation by 6-MP than did lymphocytes from subjects with intermediate or high enzyme activities (0.529 +/- 0.068 and 0.327 +/- 0.064 microM, respectively, P less than .05 for both comparisons, n = 3 in both cases).(ABSTRACT TRUNCATED AT 250 WORDS)