Recent data indicate that NAD(P)H: quinone oxidoreductase 1 (NQO1) is important in the activation of mitomycin C. A polymorphism in human NQO1 (609C>T) is associated with diminished NQO1 activity. The purpose of our study was to determine the effect of the 609C>T polymorphism on tumor NQO1 activity and overall survival in patients with disseminated peritoneal cancer receiving intraperitoneal mitomycin C therapy. Patients with disseminated peritoneal cancer of gastrointestinal or other origin were eligible. Following aggressive surgical debulking, patients were administered a 2-h heated (40.5°C) intraperitoneal perfusion with mitomycin C. NQO1 activity was determined in tumor tissue obtained during surgery and patients were genotyped for the NQO1 C609T polymorphism using a polymerase chain reaction-based method. The major response variable monitored during the trial was overall survival. Of the 117 patients genotyped for the NQO1 609C>T polymorphism, 67% were wild-type (WT), 31% were heterozygous (HE), and 2% were homozygous mutant (HM). In tumor tissue, the mean NQO1 activities from WT (n= 14) and HE (n= 5) patients were 794±603 and 70±133.1 nmol/min/mg protein respectively (P= 0.006). Significant differences in survival between WT versus HE/HM genotypes were noted in optimally debulked patients (R0/R1) (43.6+ months, median not yet reached versus 23 months respectively,P= 0.037) and in patients with peritoneal carcinomatosis of colonic origin (18.2 versus 11.5 months respectively,P= 0.050). These data indicate that the NQO1 609C>T polymorphism results in significantly reduced tumor NQO1 activity and reduced survival in subsets of patients receiving intraperitoneal hyperthermic mitomycin C therapy.