2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed during the cooking of meats and poultry. PhIP is a carcinogen in rodents and a potential human carcinogen. Several short-term biomarkers of PhIP have been established for human biomonitoring, but validated long-term biomarkers of the biologically effective dose of PhIP remain to be developed. Metabolites of PhIP have been reported to covalently bind to human serum albumin (SA), which is the most abundant protein in plasma; however, the chemical structures of PhIP-SA adducts are unknown. Cysteine 34 is one of 35 conserved Cys residues in SA across species. Thirty-four of these Cys are involved in 17 disulfide bonds. The single unpaired Cys 34 residue in SA is well-known to react with carcinogenic metabolites and toxic electrophiles. 2-Nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO 2-PhIP), 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), and 2-nitroso-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO-PhIP), three genotoxic metabolites of PhIP, were reacted with purified human SA or human plasma, and the SA adduction products, following enzymatic digestion, were separated by ultra performance liquid chromatography and characterized with a linear quadrupole ion trap mass spectrometer. The major adduct of NO 2-PhIP was formed at the Cys 34 of SA with bond formation occurring between the sulfhydryl group of Cys and the C-2 imidazole atom of PhIP. The major adducts formed between SA and HNOH-PhIP or NO-PhIP were identified as acid-labile sulfinamide linkages at Cys 34. These PhIP-SA adducts represent a measure of bioactivation of PhIP and may serve as long-term biomarkers of the biologically effective dose of PhIP.