Excision repair of DNA damage has been analyzed in primary and established cell cultures of Drosophila melanogaster. Chemical and enzymatic assays for pyrimidine dimers reveal a strong deficiency in dimer excision from cells which are mutant at the mei-9 locus. Single-strand interruptions, which appear in high molecular weight DNA after ultraviolet irradiation of control cells have been monitored by alkaline elution. The appearance of such breaks is greatly enhanced by inhibitors of DNA synthesis. In mutant mei-9D2 cells, on the other hand, the level of ultraviolet-induced breaks is much reduced and inhibitors fail to potentiate the response. These results imply that the inhibitors cause an accumulation of the transient strand interruptions that normally occur in excision repair by reducing the rate of the resynthesis step. Failure of the mei-9D2 cells to accumulate such intermediates strongly suggests that the initial nicking never occurs in these cells. Confirmatory experiments have also been performed with the alternate mutagen N-acetoxy-N-acetyl-2-aminofluorene.