Background: The enormous sequence diversity of HIV-1 has been a major obstacle in the development of a globally useful vaccine for AIDS. The consensus and ancestral sequence-based immunogens minimize the genetic distance between contemporary isolates and vaccine strains. Hence these sequences may be promising candidates for HIV vaccines or serve as a universal reagent set for evaluating Gag-specific responses. Methods: In this study, we measured the T-cell reactivity to consensus (subtype A, B, C and group M), ancestral (group M and subtype B) and HXB2 Gag peptides (15-mers overlapping by 11) in HIV-1-infected subjects from two reference populations. We evaluated the Gag-specific T-cell responses in 43 chronically infected US (subtype B) and 13 Zambian (subtype C) subjects using an interferon-γ enzyme-linked immuno-sorbent spot assay. Results: Our findings demonstrate a broad cross-reactivity of nearly 70% among all the seven Gag immunogens evaluated. Consensus M sequences elicited similar levels of responses as did the consensus B, ancestral subtype B and HXB2 peptides in subtype B-infected US patients. In subtype C-infected Zambian subjects, responses of similar breadth and magnitude were elicited by consensus C, consensus M and ancestral M peptides. Conclusion: Our data demonstrate that peptide pools based on consensus or ancestral M-based sequences can be used to evaluate Gag-specific responses elicited by subtype B or subtype C-based immunogens. © 2006 Lippincott Williams & Wilkins.