A hotspot for mutagenesis by N-acetyl-2-aminofluorene (AAF) was site-specifically modified with 2-aminofluorene (AF) and AAF adducts, and the mutation frequencies and specificities were determined and compared. Previous work has shown that the presence of an AAF adduct in a NarI sequence (GGCGCC) results a high mutation frequency for a CG double base pair deletion. In the present study, an M13 derivative was constructed that contained a NarI recognition sequence in the beta-galactosidase gene of bacteriophage M13mp9. This derivative was site-specifically modified with either an AF or an AAF adduct, the products were characterized, and these templates were then transformed into Escherichia coli wild-type strain JM103 or uvrA strain SMH12. The levels and mutation spectra were determined either with or without SOS induction. It was found that, with SOS functions induced, the measured mutation frequencies were substantially higher in all cases. More importantly, the types of mutations induced by the AAF and AF adducts were very different: AAF adducts induced almost exclusively CG double base deletion mutations, whereas AF adducts gave rise specifically to base-substitution mutations. The AF-derived mutation spectrum included both G to T and G to A mutations. The results are discussed in light of the current views on the relationship between the DNA structure and mutagenesis.