Repair of the exocyclic DNA adduct propanodeoxyguanosine (PdG) was assessed in both in vivo and in vitro assays. PdG was site-specifically incorporated at position 6256 of M13MB102 DNA, and the adducted viral genome was electroporated into repair-proficient and repair-deficient Escherichia coli strains. Comparable frequencies of PdG --> T and PdG --> A mutations at position 6256 were detected following replication of the adducted genomes in wild-type E. coli strains. A 4-fold increase in the frequencies of transversions and transitions was observed in E. coli strains deficient in Uvr(A)BC-dependent nucleotide excision repair. A similar increase in the replication of the adduct containing strand was observed in the repair-deficient strains. No change in the frequency of targeted mutations was observed in strains deficient in one or both of the genes coding for 3-methyladenine glycosylase. Incubation of purified E. coli Uvr(A)BC proteins with a duplex 156-mer containing a single PdG adduct resulted in removal of a 12-base oligonucleotide containing the adduct. Incubation of the same adducted duplex with Chinese hamster ovary cell-free extracts also resulted in removal of the adduct. PdG was a better substrate for repair by the mammalian nucleotide excision repair complex than the bacterial repair complex and was approximately equal to a thymine-thymine dimer as a substrate for the former. The results of these in vivo and in vitro experiments indicate that PdG, a homolog of several endogenously produced DNA adducts, is repaired by the nucleotide excision repair pathway.