hMLH1 is involved in DNA mismatch repair and its defects cause hereditary non-polyposis colorectal cancer (HNPCC) as well as other types of cancer. A defective DNA mismatch repair system results in genetic instability, also referred to as microsatellite instability (MSI), which is a good indicator of HNPCC. Using in silico analysis of oligo-capping cDNA sequences, we initially identified a splicing (variant type 2) whose second exon is 5 bp shorter than that of a genuine hMLH1 transcript (variant type 1) and a transcript using alternative promoter (variant type 3) whose transcription starts about 300 bases downstream of variant type 1. We then compared the expression level of variant types 1 and 3 among six colorectal cancer cell lines using real-time PCR. As a result, we found that the cell lines that completely suppress the expression of variant type 1 by hypermethylation expressed variant type 3 to a certain extent. This result suggests that the expression of variant types 1 and 3 did not completely follow the same transcription mechanism. We also found that the cell lines showing MSI to be positive either expressed variant type 3 more than type 1 or expressed only variant type 3. These results showed the potential applicability of mRNA expression analysis to molecular diagnostic tests of MSI-positive cancer types.