BCNU was reported to have about a 6- to 8- fold lower cytotoxic potency than ACNU in cell lines naturally deficient in O6-AGT. In seven tumor cell lines with an O6-AGT activity ranging from 40 to 360 fmol/mg the cytotoxic potency of BCNU, ACNU and HeCNU, without and after O6-AGT depletion by O6-BG, was determined. Without O6-AGT depletion, BCNU was superior to both other drugs in tumor cells with high O6-AGT activity. After O6-AGT depletion, the cytotoxic potency (comparison of IC50 values) of ACNU was higher than that of BCNU (p=0.016) or that of HeCNU (p=0.016) in all tumor cell lines. We conclude that (without O6-AGT depletion) BCNU is the drug of choice especially in tumor cells with high transferase activity. The higher cytotoxic potency of ACNU after O6-AGT depletion as compared to BCNU after O6-AGT depletion is countered by the higher toxicity of ACNU in patients necessitating a clinical dose reduction as compared to BCNU. Thus, we would not expect superiority of ACNU + O6-BG over BCNU+ O6-BG after systemic administration.