Fried beef and commercial beef extract contain Ames/Salmonellaframeshift mutagens that require microsomal (S‐9) activation. To ascertain which fraction(s) of beef muscle contain(s) the essential precursors, aqueous (1:1) homogenates of lean round steak were centrifuged to give an insoluble residue1(R1) and a soluble supernatant1(S1). S1was then boiled for 30 min and again centrifuged, yielding a residue2(R2) and a soluble supernatant2(S2). S2represents only 5% of the meat dry wt and it contains only 10* of the H2O‐soluble protein, but it contributes all of the S‐9 dependentSalmonellaTA1538 mutagenic activity in boiled homogenates. Mutagen formation from S2boiled for 0–30 h at a constant volume increases exponentially with time and displays sharp pH optima at 4.0 and 9.0. By molecular ultrafiltration the pH 4.0 mutagen precursors in S2have molecular wts < 500. They are also stable to lyophilization. These observations and the disappearance of certain amino acids upon boiling at pH 4.0 suggest that select amino acids may be reactants for low‐temperature mutagen formation.