AbstractMethionine aminopeptidases with a universal specificity have been revealed from the sequences of the amino‐terminal region of mutant forms of yeast iso‐1‐cytochrome c and from a systematic examination of the literature for amino‐terminal sequences formed at initiation sites. The aminopeptidase removes amino‐terminal residues of methionine when they precede certain amino acids, with a specificity that appears to be determined mainly by the residue adjacent to the methionine residue at the amino terminus. The result with the mutationally altered iso‐1‐cytochromes c and the results from published sequences of other proteins from a wide range of prokaryotes and eukaryotes suggest that the aminopeptidase usually cleaves amino‐terminal methionine when it precedes residues of alanine, cysteine, glycine, proline, serine, threonine and valine but not when it precedes residues of arginine, asparagine, aspartic acid, glutamine, glutamic acid, isoleucine, leucine, lysine or methionine. We suggest that the specificity is almost always determined simply by the size of the side chain of the penultimate residue; methionine is usually cleaved from residues with a side chain having a radius of gyration of 1·29 Å or less, but is not cleaved from residues with larger side chains.