Three alleles of the human reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase-1 (NQO1) gene are known; wild-type, 609C>T variant, and 465C>T variant; designated as NQO1*1 and NQO1*2, and NQO1*3, respectively. Previously, we found NQO1*3 in one allele of HCT-116 cells, and in both alleles of the mitomycin C-resistant subline, HCT-116R30A. The NQO1 protein content of HCT-116R30A is 5% that of HCT-116. RT-PCR revealed an exon-4-deleted NQO1 mRNA in both cell lines indicating alternative splicing. However, the cause of the lower expression of NQO1 in HCT-116R30A is unknown. Current data show that HCT-116R30A cells are able to express NQO1 protein from transfected cDNA constructs when RNA splicing is omitted. The ratio of full-length to exon-4-deleted mRNA measured by semiquantitative PCR shows that HCT-116 and HCT-116R30A have ratios of 64 : 36 and 34 : 66, respectively. All other cell lines tested have a ratio of 90 : 10, including HT-29, NIH-125 and NCI-H1688 (homozygous NQO1*1); MCF-7 and HL-60 (heterozygous NQO1*1/*2); and MDA-MB231 (homozygous NQO1*2/*2). Alternative splicing of NQO1 at the 5'-splice site of intron-4 increased in cells with NQO1*3. The 465C>T single nucleotide polymorphism (SNP) disrupts the consensus sequence at the 5'-splice site, which is required for binding by U1 small nuclear RNA (U1 snRNA) in spliceosomes. This defective RNA splicing was partially corrected by transfecting HCT-116R30A cells with U1 snRNA constructs, containing base changes to compensate for the 465 SNP. NQO1 protein and enzymatic activity increased with corrected splicing. The 465 SNP was the major cause of increased alternative splicing and decreased expression of NQO1 protein in HCT-116R30A cells.