Minor histocompatibility peptides (mHps) derived from polymorphic segments of endogenous proteins are thought to be targets for graft-versus-host and graft-versus-leukemia reactions after HLA-identical stem cell transplantation. A great majority of antigenic peptides is generated by fragmentation of proteins in the course of proteasomal processing. An algorithm was recently developed to predict cleavage sites during proteasomal processing. We tested the accuracy of the algorithm to predict mHps using 18 amino acid (AA) sequences of minor histocompatibility antigens (mHags) encoded by autosomal genes representing single nucleotide polymorphisms or by Y-chromosomal genes. The algorithm correctly predicted the C-termini of 11 of 13 experimentally confirmed mHps: 1) Correct prediction of C- and N-termini, e.g., for HA-1H; 2) Correct prediction of C- and N-termini while anticipating intra-epitope cleavage sites, e.g., for SMCY-A*0201; 3) Correct prediction of C-termini and N-terminal extensions, e.g., for HA-8R/V; and 4) Correct prediction of C-termini and N-terminal extensions while anticipating intra-epitope cleavage sites, e.g., for UTY-B8. Analysis of experimentally unconfirmed allelic counterparts of four autosomal mHags showed that AA substitutions either led to the insertion of an epitope-destroying cleavage site (e.g., in HA-1R) or abolished the correct C-terminus (e.g., in HA-2M). The proteasomal processing algorithm provides reliable data on the generation of mHps and forecasts their presence or absence. Combined with MHC class I ligand prediction, it can be a useful tool for the prediction of generation and presentation of new CTL epitopes derived from minor histocompatibility antigens.