Nucleotide excision repair (NER) was measured in human cell extracts incubated with either supercoiled or linearized damaged plasmid DNA as repair substrate. NER, as quantified by the extent of repair synthesis activity, was reduced by up to 80% in the case of linearized plasmid DNA when compared with supercoiled DNA. An excess of undamaged linearized plasmid in the repair mixture did not interfere with DNA repair synthesis activity on a supercoiled damaged plasmid, indicating a cis-acting inhibiting effect. In contrast, gaps on circular or linearized plasmids were filled in identically by the DNA polymerases operating in the extracts. When the extent of damage-dependent incision activity was measured, a approximately 70% reduction of repair incision activity by human cell extract was observed on linearized damaged plasmids. Recessed, protruding, or blunt ends were similarly inhibitory. NER activity was partly restored when the extracts were preincubated with autoimmune human sera containing antibodies against the nuclear DNA end-binding heterodimer Ku. In addition, the inhibition of repair activity on linear damaged plasmids was released in extracts from rodent cells deficient in Ku activity but not in extracts from murine scid cells devoid of Ku-associated DNA-dependent kinase activity.