Viral culture (VC), polymerase chain reaction (PCR) and in vitro antibody production (IVAP) by peripheral blood mononuclear cells were compared for the early diagnosis of HIV-1 infection in 46 infants born to HIV-1 seropositive mothers. The ten children considered infected on the basis of clinical signs and persistence of anti-HIV-1 antibodies had at least one positive viral culture and seven were always positive in both PCR and IVAP tests. PCR and IVAP tests were occasionally negative in three infected children. Among 30 healthy children who became seronegative and were always negative for viral culture, 22 (73.3%) were also repeatedly negative in PCR and IVAP. We report 6 cases of children classified as P2A at the term of this study but who had lost anti-HIV-1 antibodies. They presented at least one positive viral culture and occasional positive PCR and/or IVAP results. The results indicate that the combination of viral culture, PCR and IVAP tests improves the early diagnosis of pediatric HIV infection.