A whole cell extract prepared from Schizosaccharomyces pombe was shown to be active in an assay for repair of plasmid DNA damaged by either ultraviolet (UV) light or gamma-radiation. The assay allows for analysis of repair synthesis at single-strand nicks generated by gamma-rays and analysis of the incision step and repair synthesis in UV-light-damaged DNA. Repair synthesis of DNA damaged by either UV light or gamma-rays was shown to depend on the presence of ATP in the reaction mixture. However, incision at pyrimidine dimers did not require the addition of exogenous ATP. These studies showed that plasmid DNA containing a single pyrimidine dimer or one single-strand nick is a suitable substrate in this assay system. S. pombe is a genetically well-defined eukaryotic organism and many radiation-sensitive mutant derivatives have already been described, making this a powerful system in which to study DNA excision repair.