We have devised a method to evaluate the capacity of mammalian cell extracts to incise damaged DNA in vitro. The assay uses damaged-plasmid DNA as a substrate for nucleotide excision repair by cell extracts. During this process, enzymatic incision of the damaged DNA is followed by DNA resynthesis. Under our assay conditions, the DNA synthesis stage of excision repair is prevented by limiting dNTP concentration and including the specific DNA polymerase inhibitor aphidicolin. Incisions are quantitatively detected by [alpha-32P]dAMP incorporation catalysed by the Klenow fragment of E. coli DNA pol I at nicked sites in plasmids purified from incision reactions. Lesion-specific incision is an ATP-dependent process; it was observed in plasmids modified with three different DNA damaging agents and damage-dependent incisions were abolished with extracts from xeroderma pigmentosum excision-repair deficient cell lines, indicating that this in vitro incision assay is dealing with true nucleotide excision repair.