A 16mer oligonucleotide containing a single guanine residue at nucleotide 13 from the 3' end was treated with the (+)-enantiomer of the 7,8-dihydrodiol 9,10-epoxide of benzo[a]pyrene (B[a]P). Oligonucleotides containing either an adduct in which the epoxide ring was opened trans or cis by the amino group of the guanine residue were separated by chromatography and identified by 32P postlabeling and circular dichroism spectroscopy. In the presence of nucleotide triphosphates and DNA polymerase (either Sequenase, version 2.0 or human polymerase alpha), it was found that the B[a]P adducts inhibited extension of an 11mer primer opposite the nucleotide 3' to the adduct in the template. Under various conditions, this inhibition was greater for the cis adduct than for the trans adduct. After a 10 min incubation with Sequenase, primer extension was reduced to approximately 20% of that seen with unmodified oligonucleotide by the trans adduct and was almost completely inhibited by the cis adduct. When a 12mer primer was used to examine nucleotide incorporation directly across from the guanine or adducted guanine residues, it was clear that deoxycytidylic acid was preferentially incorporated in all cases but that the incorporation was severely inhibited by both the cis and trans adducts. These findings suggest that a cis adduct is a more effective block to replication than a trans adduct, and that these adducts may not be very efficient mutagenic lesions.