We studied the reactions with DNA of α-acetoxy-N-nitrosopyrrolidine (α-acetoxyNPYR) and α-acetoxy-N-nitrosopiperidine (α-acetoxyNPIP) in order to obtain more information on adduct formation by metabolic activation via α-hydroxylation of two cyclic nitrosamines, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP). Enzyme hydrolysis and HPLC analysis of DNA that had been reacted with unlabeled, [14C]-, or [3H]α-acetoxyNPYR permitted the positive identification of N2-(tetrahydrofuran-2-yl)deoxyguanosine (THF-dG). It was identified by comparison of its UV spectrum and retention time to those of a standard, by conversion upon NaBH4 treatment to N2-(4-hydroxybutyl)deoxyguanosine, and by neutral thermal hydrolysis to 2-hydroxytetrahydrofuran (THF-OH). The levels of THF-dG in DNA exceeded that of other adducts of α-acetoxyNPYR. Reaction of α-acetoxyNPIP with DNA followed by enzyme hydrolysis and HPLC analysis resulted in the positive identification of two diastereomers of N2-(3,4,5,6-tetrahydro-2H-pyran-2-yl)deoxyguanosine (THP-dG) by comparison of their retention times and UV spectra to those of standards. The levels of THP-dG were similar to those of THF-dG formed from α-acetoxyNPYR. Neutral thermal hydrolysis of DNA that had been reacted with α-acetoxyNPIP produced 2-hydroxy-3,4,5,6-tetrahydro-2H-pyran (THP-OH). Studies on the mechanism of formation of THF-dG and THP-dG indicated that stable cyclic oxonium ion-derived electrophiles could be their major precursors. Our data provide the first evidence for the formation of cyclic oxonium ion-derived DNA adducts from cyclic nitrosamines and indicate some potential differences in DNA binding between α-acetoxyNPYR and α-acetoxyNPIP. THF-OH and THP-OH released from DNA by neutral thermal hydrolysis may be useful as dosimeters of adduct formation by NPYR and NPIP.