Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type 1 (HIV-1), mostly to prevent mother-to-child HIV transmission in developing countries. One of the limitations of nevirapine use is severe hepatotoxicity, which raises concerns about its administration, particularly in the perinatal and pediatric settings. Nevirapine metabolism involves oxidation of the 4-methyl substituent to 12-hydroxy-NVP and the formation of phenolic derivatives. Further metabolism of 12-hydroxy-NVP by phase II esterification may produce electrophilic derivatives capable of reacting with DNA to yield covalent adducts, which could potentially be involved in the initiation of mutagenic and carcinogenic events. In the present study, we have investigated the reactivity of the synthetic model electrophile, 12-mesyloxy-NVP, toward 2'-deoxynucleosides and DNA. Parallel synthetic studies were conducted with 12-bromo-NVP and 3',5'- O-bis( tert-butyldimethylsilyl)-2'-deoxynucleosides, using palladium(0) catalysis. Multiple adducts from deoxyguanosine, deoxyadenosine, and deoxycytidine were isolated in the reactions with 12-mesyloxy-NVP and characterized by NMR and MS. The adduct structures consistently involved binding through C12 of NVP and N7 or N9 of deoxyguanosine; N1, N3, or N9 of deoxyadenosine; and N3 of deoxycytidine. Reactions conducted under palladium(0) catalysis yielded adducts through O (6) and N1 of deoxyguanosine, N1 of deoxyadenosine, and N3 of deoxycytidine. At least seven deoxynucleoside-NVP adducts were present in DNA reacted with 12-mesyloxy-NVP and enzymatically hydrolyzed. Four of these adducts were identified as 12-(deoxyadenosin-N1-yl)nevirapine, 12-(deoxycytidin-N3-yl)nevirapine, 12-(deoxyguanosin- O(6)-yl)nevirapine, and 12-(deoxyadenosin- N(6)-yl)nevirapine. One depurinating adduct, 12-(guanin-N7-yl)nevirapine, was identified in the thermal neutral DNA hydrolysate. If formed in vivo, some of these adducts would have considerable mutagenic potential. Our results thus suggest that NVP metabolism to 12-hydroxy-NVP may be a factor in NVP hepatocarcinogenicity.