Fried beef and commercial beef extracts contain Ames/Salmonellaframeshift mutagens that form at relatively low temperatures (100–200°C). To investigate the types of natural components in beef muscle that give rise to frameshift mutagenic activity, we previously devised a model boiling system and demonstrated that all of theSalmonellaTA1538 activity is formed from H2O‐soluble, < 500 molecular wt compounds that are present in round steak supernatant fractions (S1and S2). S2is derived from S1, the soluble fraction of homogenized beef, by a brief 30 min boil and centrifugation. We now report that proteolysis of beef S1with papain, trypsin, or chymotrypsin (± carboxypeptidase A) increases the mutagenic activity of boiled S2by 1.8–4.5 fold over the baseline range of 90–100 TA1538 revertants/108bacteria/g dry beef/14 h at pH 4.0. Sequential treatment of beef S1with chymotrypsin followed by carboxypeptidase A is the most efficient mutagen enhancing digestion (415 revertants/g dry beef or7.7revertants/mg of S2soluble protein). Fourteen h boiled, proteolytic digests of round steak insoluble‐protein fraction (R1), soybean protein, bovine serum albumin, and bovine α‐lactalbumin contain much less TA1538 activity/mg of soluble protein than beef S2(0.01–1.5 versus7.4revertants/108bacteria). For comparison, S2fractions prepared from chicken (light meat), chicken (dark meat), pork chops, turkey (light meat), beef liver, fish (sole), and whole eggs yield 89, 60, 71, 67, 58, 36, and < 1 TA1538 revertants/108bacteria/g of initial dry wt. Despite their low TA1538 activity yields, S2‐like fractions from proteolyzed beef R1, soybean protein, and the two pure albumins, and S2fractions from sole and eggs were comparable to our standard beef S1or S2in their amino acid compositions. Collectively, these mutagenicity and amino acid composition data indicate that select amino acids and/or small peptides plus some essential non‐amino acid compounds are the mutagen precursors in beef muscle.