To define the epitopes present within the V3 loop sequence recognized by five HIV-1 envelope-specific T-cell lines, a panel of V3 LAI peptides bearing sequential truncations from both the N- and C-terminus was synthesized and tested for their ability to induce proliferation. Each individual T-cell line had a different pattern of response against the truncated V3 peptides, demonstrating the presence of a cluster of CD4+ T-cell epitopes within the V3 loop. To assess the ability of these envelope-specific T-cell lines to recognize and proliferate in response to V3 loops of different viral strains, they were tested against a panel of heterologous V3 loop peptides derived from different viral genotypes within and outside of HIV-1 clade B. There was no proliferative response against heterologous V3 loops by any of the lines, demonstrating that recognition of the V3 epitopes is highly strain specific. One of the defined epitopes was shown to elicit a cytotoxic response as well, suggesting the multifaceted role that the CD4+ T cell might play in HIV-1 disease.