Heterocyclic amines produced from overcooked foods are extremely mutagenic in numerous in vitro and in vivo test systems. One of these mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), induces breast tumors in rats and has been implicated in dietary epidemiology studies as raising the risk of breast cancer in humans. Efforts in our laboratory and others have centered on defining the exposure to PhIP and other dietary mutagens derived from cooked food. We accomplish this by analyzing the foods with a series of solid-phase extractions and HPLC. We have developed an LC/MS/MS method to analyze the four major human PhIP metabolites (sulfates and glucuronides) following a single meal containing 27 microg of cooking-produced PhIP in 200 g of grilled meat. Although the intake of PhIP was similar for each of eight women, the total amount excreted in the urine and the metabolite profiles differed among the subjects. It appears that adsorption (digestion) from the meat matrix, other foods in the diet, and genetic differences in metabolism may contribute to the variation. The four major metabolites that can be routinely assayed in the urine are N(2)-OH-PhIP-N(2)-glucuronide, PhIP-N(2)-glucuronide, 4'-PhIP-glucuronide, and N(2)-OH-PhIP-N3-glucuronide. This work is suited to investigate individual exposure and risk, especially for breast cancer, from these potent dietary mutagens.