The N-acetylation of 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2) and other heterocyclic aromatic amine promutagens isolated originally from protein pyrolysates was investigated using liver cytosols from various animal species and acetyl-CoA as an acetyl donor. Marked species differences in the enzymatic N-acetylation activity of these heterocyclic amines were observed. The N-acetylation activity also varied among the substrates used. The N-acetylation of these heterocyclic amines by hepatic cytosols from all animal species used was much less than that of the non-heterocyclic aromatic amine carcinogen, 2-aminofluorene (2-AF): the N-acetylation of Trp-P-2 was more than one hundred times less than that of 2-AF. These results suggested that the metabolic activation pathway of these mutagenic heterocyclic amines is different from that of 2-AF. The hamster but not rat cytosol showed the ability to utilize N-hydroxy-2-acetylaminofluorene and 2-acetylaminofluorene as acetyl donor in the N-acetylation of Trp-P-2.