The associations among timing of maternal-fetal human immunodeficiency virus (HIV) transmission, infant age at first positive HIV culture, and CD8+ lymphocyte activation were examined for 74 perinatally infected infants. Nineteen of the infected infants had positive HIV cultures at < or =7 days of life, and 55 had negative HIV cultures at < or =7 days but were positive later. Of the infants with early positive HIV-1 cultures, 15 of the 17 tested with DNA polymerase chain reaction methods had concordant results. The percentage of CD8+ and HLA-DR+ lymphocytes (CD8+DR+%) during the first week of life was significantly higher in infants with early compared with late positive cultures (median CD8+DR+% of 5.0% versus 2.0%, p = 0.0006). The CD8+DR+% was similar between uninfected infants and infants with late positive cultures during the first week of life (median 2%) but increased in infants with late positive cultures to 6% by 1 month. The CD4+% during the first 6 months of life was not different between infants with early or with late positive cultures, but infants with the highest CD8+DR+% at < or =7 days of life had significantly lower CD4+% at < or =7 days and at 1, 2, and 4 months of age. These data show that early CD8+ lymphocyte activation is associated with early positive HIV cultures and lower CD4+ percentages during early infancy and are consistent with the hypothesis that early positive cultures positivity may indicate in utero HIV infection.