The metabolism of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), was examined in rat pancreatic microsomes. No pyridine N-oxidation or α-hydroxylation were observed in these preparations. However, one unidentified metabolite of NNAL (unknown A) and two unknown metabolites of NNK (unknowns B and C) were formed. These metabolites were also detected in rat liver microsomal incubations of NNK and NNAL. Studies using [5-3H]-NNK and [Me-3H]NNK demonstrated that the metabolites contained both the pyridyl and methyl portions of the parent compound. Similar results were obtained with NNAL. Formation of unknown C required active microsomes, NADP+, and an NADPH regenerating system. The regenerating system was not required for the formation of NNAL unknown metabolite A or NNK unknown metabolite B. Chemical characterization of unknowns A and B by NMR, UV, and electrospray ionization MS demonstrated that they are NADP+ analogs in which the nicotinamide portion has been replaced by NNAL or NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol adenosine dinucleotide phosphate [(NNAL)ADP+] and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone adenosine dinucleotide phosphate [(NNK)ADP+]. Unknown C was identified as (NNK)ADPH. Both (NNK)ADP+ and (NNK)ADPH were formed from NNK while only (NNAL)ADP+ was produced from NNAL. These NADP+ derivatives were also formed when porcine brain NAD+ glycohydrolase was incubated with NADP+ and NNK or NNAL. These results indicate that NNK and NNAL are substrates for rat liver and pancreatic microsomal NAD+ glycohydrolase-catalyzed transglycosylation reactions.