Identifying immunodominant regions of HIV-1 that are recognized by CD8+ T lymphocytes in infected individuals may be important for the design and evaluation of candidate HIV-1 vaccines, particularly for developing countries. In this study, cryopreserved peripheral blood mononuclear cells (PBMCs) from 15 chronically HIV-1-infected U.S. volunteers were screened for HIV-1 Gag-specific T lymphocyte interferon γ production in an enzyme-linked immunospot (ELISpot) assay matrix format, using overlapping HIV-1 subtype A, B, and C Gag peptide pools. In the initial matrix screen, responses to HIV-1 subtype B Gag were detected in 11 of 15 (73%) of seropositive individuals and in none of 4 HIV-1-seronegative controls. There were differences in both the breadth and magnitudes of the responses observed in the matrix assay. Responses varied in breadth, ranging from broad responses (more than four peptides) of moderate magnitude (<100 spot-forming cells [SFCs]/105 PBMCs) to narrowly focused (two or fewer peptides), more potent responses (>150 SFC/105 PBMCs). Responses to A, B, and C clade peptides of HIV-1 Gag revealed that all responders to subtype B peptides were also found to recognize corresponding peptides from at least one of the other clades. The ability to recognize cross-clade peptides with one or two amino acid substitutions relative to the B clade peptide was both peptide and patient dependent. Overall, our results show that the ELISpot matrix algorithm described here may be an efficient approach for characterizing cross-clade CD8+ T cell responses in either seropositive individuals or in seronegative HIV-1 vaccine recipients.