Alloreactive mouse cytotoxic T lymphocytes (CTL) specific for particular peptides presented by H-2K(b) molecules on mouse cells were found to recognize human cells transfected with K(b). The CTL-recognized peptides (probably derived from conserved proteins) were extracted from K(b)-expressing human or mouse cells, respectively, and compared biochemically by high resolution high performance liquid chromatography. The results strongly suggest identity of peptides processed by cells from both species and thus indicate that the specificity of the processing machinery used in the major histocompatibility complex (MHC) class I-restricted pathway, probably including enzymes, transport mechanisms, and chaperons, is highly conserved across species. The results are consistent with the notion that MHC molecules themselves have an instructive role in processing.