N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen that is present in the diet and tobacco smoke and may form endegenously in humans. Biomarkers to assess NPYR exposure and metabolic activation in humans are needed. The cyclic N7,C-8 guanine adduct 2-amino-6,7,8,9-tetrahydro-9- hydroxypyrido[2,1-f]purin-4(3H)-one (8), which is formed in tissues of rats treated with NPYR, is one potential candidate for such a biomarker. In this study, we evaluated the formation of this and other NPYR adducts in reactions of α-acetoxyNPYR with dGuo, Guo, DNA, and RNA and determined the extent of urinary excretion of adduct 8 in rats treated with NPYR. α-AcetoxyNPYR, a stable precursor to the major product of NPYR metabolic activation, was allowed to react with dGuo, Guo, DNA, or RNA at 37 °C, pH 7. The most striking observation was that the cyclic N7,C-8 guanine adduct 8 was formed 9 times more extensively in the reaction with Guo than with dGuo. It was also formed 2.5 times more extensively in RNA than in DNA. In rats treated with NPYR, levels of the cyclic N7,C-8 guanine adduct 8 were 2 times as high in RNA than in DNA. Rats treated with [14C]adduct 8 excreted 51% of this adduct unchanged in urine. Rats treated with [3,4-3H]NPYR excreted 0.000 04% of the dose as adduct 8. The major differences in product formation in reactions of α-acetoxyNPYR with dGuo versus Guo are unusual for alkylating agents; potential mechanisms are discussed. The higher levels of adduct 8 in RNA than in DNA suggest that RNA may be superior as a source of adduct 8 as a biomarker.