The suspected major mutagenic adduct of benzo[a]pyrene, (+)-anti-B[a]P-N2-Gua, is built into the unique PstI recognition site of the Escherichia coli plasmid, pUC19, in order to study its mutagenic potential. The adduct can either be at G437, which is replicated during leading strand DNA synthesis, or at G438, which is replicated during lagging strand DNA synthesis. The DNA strand complementary to the strand containing the (+)-anti-B[a]P-N2-Gua adduct is saturated with UV lesions to minimize its potential to generate progeny. Although all in-frame mutations could have been detected, a G437----T transversion mutation is virtually exclusively obtained at a frequency of approximately 0.04% per adduct following transformation into Uvr+ E. coli when SOS is not induced, and approximately 0.18% when SOS is induced. The mutation frequency of the adduct in a Uvr- background is estimated to be approximately 0.2% when SOS is not induced, and approximately 0.9% when SOS is induced. The absence of G438----T mutations is rationalized. G----T mutations from (+)-anti-B[a]P-N2-Gua are compared to the mutational specificity of the ultimate mutagenic form of activated benzo[a]pyrene.