N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is a sulfotransferase responsible for biosynthesis of chondroitin sulfate E (CS-E). CS-E plays important roles in numerous biological events, such as neurite outgrowth. However, the role of GalNAc4S-6ST in tumor progression remains unknown. In the present study, we analyzed expression of GalNAc4S-6ST mRNA in colorectal cancer by combining real-time RT-PCR with in situ hybridization (ISH) using archived formalin-fixed and paraffin-embedded tissue sections. In 57.5% of 40 patients, expression of GalNAc4S-6ST mRNA was increased in cancer tissues compared with paired normal mucosa. ISH using an RNA probe specific for GalNAc4S-6ST revealed that it was expressed in colorectal cancer cells. Analysis of the relationship between expression of GalNAc4S-6ST as determined by real-time RT-PCR assay and various clinicopathological variables revealed that GalNAc4S-6ST was associated with vessel invasion, although a statistically significant difference was not seen (P= 0.125 for lymphatic vessel invasion and P= 0.242 for venous invasion). Taken together, we show that real-time RT-PCR combined with ISH is useful to investigate quantitatively GalNAc4S-6ST mRNA expression in formalin-fixed and paraffin-embedded tissue sections, and that GalNAc4S-6ST expressed by colorectal cancer cells plays a minor role in tumor progression.