We have investigated the proliferative response of peripheral blood mononuclear cells from individuals infected with human immunodeficiency virus type 1 (HIV-1) to synthetic peptides from the third variable loop region (V3) in the envelope protein gp120. We tested a total of 14 peptides, corresponding to 14 HIV-1 isolates belonging to four geographical locations (clades U, A, B, and D). Although differences in relative level of responses exist between individual peptides and patients, the proliferation in response to all 14 V3 peptides was significantly greater than that to unrelated control peptides. Additionally, we observed that proliferative responses of blood cells from the 10 HIV-seropositive individuals studied from the clade B region to peptides from within clades U, A, B, and D were not significantly different, indicating the cross-reactive nature of the V3-specific cell-mediated immune responses. Even though the majority of patients also exhibited antibody responses against several V3 peptides, serum samples from 50% of clade B patients exhibited antibody cross-reactivity, while proliferative responses to V3 peptides from more than one clade were observed in 80% of patients. Importantly, in two patients, decreased CD4+ cell numbers, an important surrogate marker of disease progression, significantly correlated with loss of V3 peptide-specific proliferative responses but not antibody responses. These results have important implications toward evaluating the utility of V3 peptides for designing therapeutic and/or vaccine reagents against HIV-1.