The present study used a rapid and single-step method for genotyping of NAD(P)H quinone oxidoreductase (NQO1) codon 609 polymorphism using real-time polymerase chain reaction (PCR)-analysis and subsequent melting curve analysis for the analysis of allelic distribution of NQO1. The design was a case control study of 323 Caucasians with colorectal cancer and 205 healthy controls. There was no difference in the frequencies of the mutated NQO1 allele (NQO1*2): 0.190 for control individuals and 0.195 for cancer patients, respectively (P=0.947). When this allelic distribution was further compared between non-smoking and smoking colorectal cancer patients, it appeared that the frequency of the wild-type allele NQO1*1 was higher in the smoking than in the non-smoking group [Odds ratio (OR), 0.434; 95% confidence interval (CI), 0.13-1.42]. This observation may suggest a protective role of the NQO1 wild-type allele in colon cancer susceptibility of individuals exposed to NQO1-inducing chemicals.