Recent efforts at understanding the immune response generated against human immunodeficiency virus (HIV) infection have focused on cytotoxic T lymphocyte (CTL)-mediated recognition of HIV antigens. CTLs are a major immune defense mechanism and are necessary for the recovery of many viral infections. We have previously developed a method for screening synthetic peptides for the ability to induce virus-specific major histocompatibility complex-restricted CTLs in mice. Using this method, we now report the identification of peptides from the V3 region in gp120 of seven different HIV-1 strains that are capable of inducing a virus-specific CD8+ CTL response in vivo. V3 peptides from MN and SC strains of HIV-1, which are representative of typical strains found in North America and Europe, induced CTLs that exhibited cross-reactivity against a broad range of HIV-1 strains. In addition, immunization of mice with a mixture of these V3 peptides resulted in efficient CTL responses directed against the corresponding HIV-1 strains. These data, together with information in the literature describing the CTL epitope nature of V3 peptide from HIV-1 IIIB in the context of several HLA alleles, indicate the possibility of including V3 synthetic peptides as components of potential vaccines for inducing broadly cross-reactive CTL response against a diverse array of HIV-1 strains.