Using a replica-plating procedure and a 32P-NAD+ permeable cell-screening assay, we have isolated a CHO mutant, PADR-9, which displays approximately 17% of the wild-type level of poly(ADP-ribose) polymerase activity. Biochemical analysis of the mutant using activity, Western, and Northern blot techniques indicate that relative to its parent cell, the mutant's enzyme activity, antibody recognition, and mRNA levels have been reduced to approximately the same extent. These results are consistent with a mutation in the PADR-9 cell which has resulted in a reduction in enzyme synthesis due to reduced mRNA synthesis and/or stability. Relative to wild-type CHO cells, the PADR-9 mutant has increased sensitivity to killing by DNA-alkylating agents but has normal gamma-ray sensitivity. Correlation between a decrease in poly(ADP-ribose) polymerase activity and an increased sensitivity to DNA-alkylating agents suggests that poly(ADP-ribose) synthesis may be important in the repair and/or induction of DNA damage produced by these agents.