SIV-infected Indian rhesus macaques (Macaca mulatta) are an important animal model for humans infected with HIV. Understanding macaque (M. mulatta class I (Mamu)) MHC class I-peptide binding facilitates the comparison of SIV- and HIV-specific cellular immune responses. In this study, we characterized the endogenous peptide-binding properties of three Mamu-A (A*02, A*08, A*11) and three Mamu-B (B*01, B*03, B*12) class I molecules. Motif comparisons revealed that five of the six macaque class I molecules (A*02, A*08, A*11, B*01, and B*03) have peptide-binding motifs similar to those of human class I molecules. Of the 65 macaque endogenous peptide ligands that we sequenced by tandem mass spectroscopy, 5 were previously eluted from HLA class I molecules. Nonamers predominated among the individual ligands, and both the motifs and the individual ligands indicated P2, P9, and various ancillary anchors. Interestingly, peptide binding of the Mamu-A and Mamu-B molecules exhibited cross-species peptide-presentation overlap primarily with HLA-B molecules. Indeed, all of the macaque class I molecules appeared HLA-B-like in peptide presentation. Remarkably, the overlap in macaque- and HLA-peptide presentation occurred despite divergent class I peptide-binding grooves. Macaque and human class I differing by up to 42 aa (13-23%) within the alpha-1 and alpha-2 domains, including substantial divergence within specificity pockets A-F, bound the same endogenous peptide. Therefore, endogenous peptide characterization indicates that macaque class I molecules may be the functional equivalents of HLA-B molecules.