The effect of the adenosine diphosphoribosyltransferase inhibitors, the substituted benzamides, on the cytotoxicity of 6-thioguanine (6TG) was investigated. Nontoxic concentrations of benzamides potentiated the cytotoxicity of 6TG with a dose enhancement factor of 2, producing a 6-fold increase in cell killing at 10% survival. 6TG treatment did not deplete cellular NAD levels, and in the presence of 3-aminobenzamide, there was no increase in the number of 6TG-induced DNA strand breaks. To obtain potentiation of cytotoxicity, 3-aminobenzamide had to be present in late G1-S phase during the cell cycle in which 6TG is incorporated into the DNA. These data indicate that the substituted benzamides potentiate the cytotoxicity of 6TG by a mechanism independent of an inhibition of DNA repair.