In order to provide the opportunity for women delivering newborns to have human immunodeficiency virus (HIV) testing we piloted a hospital-based voluntary HIV testing program during the newborn period using the Guthrie card. During the study period 789 women were offered newborn HIV antibody testing. Test acceptance during the newborn period (61.0%) was comparable to that reported for the prenatal period (60.6%). Overall 77.4% of women were tested in the newborn period or reported being tested in the prenatal period. Prenatal test acceptance best predicted newborn HIV test acceptance (odds ratio, 3.37; 95% confidence interval, 2.40 to 4.74). When compared to HIV testing during the newborn period prenatal HIV testing is preferable because it enables the recognition of HIV infection early during pregnancy and allows the mother the option to elect zidovudine therapy and potentially prevent infection in her newborn. However, when prenatal HIV testing is not routinely made available or cannot be assured, women should be offered the opportunity to be tested during the newborn period.