To understand the cross-reactivity of antibodies directed against variable regions of human immunodeficiency virus-1 (HIV-1) envelope (Env), chimeric immunogens were prepared from different clades with modifications in variable regions, and the resulting neutralizing antibody response was evaluated. The V3-specific neutralization activity induced by a clade B immunogen was limited to clade B viruses and was blocked by a clade B V3 peptide, but not by analogous clade A or C V3 peptides. In contrast, the V3 response elicited by a clade C immunogen cross-reacted with sensitive clade B viruses. The V3 region from a clade C virus, when introduced into a clade B sequence, elicited cross-clade activity, which could be reversed by V3 peptides derived from clades A and C. Thus, the anti-V3 antibody response elicited by a clade C immunogen could cross-react with heterologous clade viruses. Additionally, we describe a V1-specific immune response that mediated neutralization limited to the homologous HIV IIIB isolate and may be partially responsible for the commonly observed strain-specific neutralization responses elicited by vaccine immunogens.