The principal neutralizing domain (PND) of HIV-1, located within the third variable region (V3) of the gp120 envelope protein, is related to the humoral and cellular immune response. We studied the V3 PND-specific antibody response in 30 children with vertically acquired HIV-1 infection by determining the antibodies that bound synthetic peptides derived from the PND of the HIV-1MN, HIV-1SF-2, HIV-1SC, HIV-1IIIB, HIV-1RF, HIV-1ELI, and HIV-1Z6 virus strains. At a standard antigen concentration, we found that most sera (90%) reacted against PNDMN peptide, but 73.3% also cross-reacted against multiple PNDs. A search for high-affinity/avidity antibodies was conducted in an antigen-limited assay; at lower peptide concentrations, cross-reactivity was restricted to PNDMN and PNDSC in 12 of 22 broadly reactive sera. Sequence analysis of the V3 region of HIV-1 isolates indicated that patients with high-affinity/avidity antibodies to PNDMN and PNDSC had a PND with an internal 12-amino acid sequence (serotype-specific domain, SSD) that was highly homologous (> 90%) with the MN and SC SSD. Broadly reactive sera with low-affinity/avidity antibodies showed a lower degree of homology with the SSD sequence of all tested viral strains. The role of anti-PND antibodies in vertical transmission was further studied in 49 children born to HIV-1-seropositive mothers. No statistical correlation emerged between V3 antibodies and HIV-1 transmission, but we found that maternal V3 antibodies were lost soon after birth. This finding may be relevant to a new serological approach to the early diagnosis of vertically transmitted HIV-1 infection.