2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed during the cooking of meats. PhIP is a potential human carcinogen: it undergoes metabolic activation to form electrophilic metabolites that bind to DNA and proteins, including serum albumin (SA). The structures of PhIP-SA adducts formed in vivo are unknown and require elucidation before PhIP protein adducts can be implemented as biomarkers in human studies. We previously examined the reaction of genotoxic N-oxidized metabolites of PhIP with human SA in vitro and identified covalent adducts formed at cysteine³⁴ (Cys³⁴); however, other adduction products were thought to occur. We have now identified adducts of PhIP formed at multiple sites of SA reacted with isotopic mixtures of electrophilic metabolites of PhIP and 2-amino-1-methyl-6-[²H₅]-phenylimidazo[4,5-b]pyridine ([²H₅]-PhIP). The metabolites used for study were 2-nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO₂-PhIP), 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), or N-acetyloxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-acetoxy-PhIP). Following proteolytic digestion, PhIP-adducted peptides were separated by ultra performance liquid chromatography and characterized by ion trap mass spectrometry, employing isotopic data-dependent scanning. Analysis of the tryptic or tryptic/chymotryptic digests of SA modified with NO₂-PhIP revealed that adduction occurred at Cys³⁴, Lys¹⁹⁵, Lys¹⁹⁹, Lys³⁵¹, Lys⁵⁴¹, Tyr¹³⁸, Tyr¹⁵⁰, Tyr⁴⁰¹, and Tyr⁴¹¹, whereas the only site of HONH-PhIP adduction was detected at Cys³⁴. N-Acetoxy-PhIP, a penultimate metabolite of PhIP that reacts with DNA to form covalent adducts, did not appear to form stable adducts with SA; instead, PhIP and 2-amino-1-methyl-6-(5-hydroxy)-phenylimidazo[4,5-b]pyridine, an aqueous reaction product of the proposed nitrenium ion of PhIP, were recovered during the proteolysis of N-acetoxy-PhIP-modified SA. Some of these SA adduction products of PhIP may be implemented in molecular epidemiology studies to assess the role of well-done cooked meat, PhIP, and the risk of cancer.