Heterocyclic aromatic amines (HCA) are formed upon frying of poultry, fish or meat and have been shown to induce tumours in rodent bioassays. We investigated the transforming activity of HCA in an in vitro assay using the M2/C3H mouse fibroblast cell line. An external metabolic activation system (rat liver homogenate) was required in order to observe any HCA-induced cytotoxic effects or cell transforming activity. Trp-P-1 and Trp-P-2 are shown to be among the most potent transforming HCA that have been detected in food, Metabolic activation of HCA has been shown to proceed via N-hydroxylation of the exocyclic amino group. Therefore, we tested 2-hydroxyamino-1-methyl6-phenylimidazo [4,5-b] pyridine (N-2-OH-PhIP) the activated metabolite of 2-anino-1-methyl-6-phenylimidazo[4,5h]pyridine. N-2-OH-PhIP proved to be one of the most powerful compounds with transforming activity observable at a concentration as low as 30 nM. Since 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant HCA formed in fried and grilled food and N-hydroxylation appears to be the predominant pathway of human metabolism, these data support the hypothesis that HCA are involved in the aetiology of human cancer. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.