CHO cells were exposed to 11 different restriction endonucleases by electroporation and their mutagenicity was measured. Nine of them have one or more recognition sites within exons of the HPRT gene, whereas the remaining two cut in introns only. The mutagenic efficiency of the various enzymes varied markedly; mutagenicity of Sau3AI was considerably higher than that of the other enzymes. Neither cytotoxicity nor mutagenicity could be related to the number or location of recognition sites within the cDNA. A total of 417 independent restriction enzyme induced mutant clones were isolated from 20 separate experiments for molecular analysis; all nine exons of the HPRT gene were analyzed by a modified multiplex deletion screening method with polymerase chain reaction (PCR) amplification. Among spontaneously arising mutants, 70.8% showed no change in PCR pattern, indicating a small scale change (point mutation), whereas partial deletions were observed in 24.7%, and total deletions in 4.5% of mutant clones. In contrast, approximately 70% of restriction enzyme induced mutants showed partial or total deletions. There was no obvious relationship between type of break (blunt versus staggered ends), and the DNA structure of the mutations induced. For partial deletions, the distribution of breakpoints within introns appeared to occur at random, and did not correlate with the mutagenicity of a given enzyme. Thus, though DNA double-strand breaks appear to be important mutagenic lesions that can induce a high frequency of deletion mutants, no specific relationship of mutagenic potential to the type of breaks, their sites within the HPRT gene or the molecular structure of the mutations induced could be identified.