Previously, we reported an association of mitomycin C resistance and a deficiency of NAD(P)H:quinone oxidoreductase (NQO1) in HCT 116-R30A cells, a subline derived from mitomycin C-sensitive HCT 116 cells. In HCT 116 cells, we found two mRNAs coding full-length cDNAs of NQO1 differing at codon 139, one with arginine (wild type), and one with tryptophan. Only the tryptophan 139 form of mRNA was detected in HCT 116-R30A cells. In addition, an exon 4 deleted mRNA of NQO1, a product of alternative splicing, was detected in both cell lines. Analysis by semiquantitative reverse transcription-PCR showed that NQO1 mRNA coding full-length cDNAs in HCT 116-R30A cells was 15% of that present in HCT 116 cells. A Mr 26,000 protein, representing the exon 4 deleted mRNA, was not detected by polyclonal anti-NQO1 in HCT 116 sublines. Recombinant plasmids of exon 4 deleted cDNA generated a Mr 26,000 protein without enzymatic activity in Escherichia coli but not in Cos7 cells. The function of exon 4 deleted mRNA is yet unknown. The rates of decay of all NQO1 mRNAs in HCT 116 and HCT 116-R30A cells were similar. DNA sequences of the promoter regions of the NQO1 gene (-837 bp) from both cell lines did not differ from each other or from the same region of the human liver NQO1 gene. Sequences of cis elements in the 837-bp region and mRNA stability could not account for the low expression of full-length mRNA in HCT 116-R30A cells. Southern blot analysis showed the size and the intensity of the NQO1 gene in the two cell lines to be similar. This result was confirmed by semiquantitative PCR analysis of a 450-bp fragment in the NQO1 gene containing codon 139 and the exon 4 region. Digestion of this PCR-amplified fragment by restriction enzyme MspI revealed that HCT 116 cells have two heterozygous NQO1 alleles, a wild-type and a tryptophan 139 form. The functional wild-type NQO1 allele was not detected in HCT 116-R30A cells. Sensitive and resistant cell lines each contained one normal and one abnormal chromosome 16. Loss of the wild-type NQO1 allele in HCT 116-R30A cells did not result from a loss of chromosome 16 or copies of the NQO1 gene. Alteration of factor(s) such as trans-acting factors and DNA methylation may be involved in the down-regulation of NQO1 in the mitomycin C-resistant HCT 116-R30A cells.