Mitochondrially synthesized polypeptides of human, monkey, and mouse cells were compared using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A single molecular weight variant, the major interspecific variant (MIV), was identified in human cells as compared to monkey and mouse. The peptide maps of MIV were compared between the three species using a two-dimensional proteolytic digest (2D-PD) gel system. A number of conserved peptides were found, indicating that the MIVs have a common function. Other MIV peptides were species specific. These results confirm the conserved nature of mitochondrial polypeptides and demonstrate the utility of 2D-PD gels in testing for protein alleles and detecting subtle protein variants.