The activity of -galactoside ␣2,6-sialyltransferase (ST6Gal.1), the enzyme responsible for the addition of sialic acid in ␣2,6-linkage to N-acetyllactosaminic (Gal1,4GlcNAc) units of glycoconjugates, is increased in the vast majority of colon cancer specimens, and a positive correlation with an invasive phenotype has been suggested by several studies. In many tissues, ST6Gal.1 is regulated mainly at the transcriptional level through the use of different cell-specific promoters which generate transcripts differing in their 5'-untranslated regions. With the aim of understanding the molecular bases of the increased ST6Gal.1 expression in colon cancer, we investigated the expression of mRNA species in colon cancer cell lines and the relationship with enzyme activity and extent of ␣2,6-sialylation of cell glycoproteins. All cell lines examined express the form containing the 5'-untranslated exons Y and Z, typical of the ''basal'' expression of the gene, while others express also the liver transcript. This indicates that colon cancer cell lines can be grouped according to expression of the liver transcript of ST6Gal.1. The cell lines expressing only the Y؉Z form display, in general, a lower activity:mRNA ratio, which might indicate reduced translational efficiency. The level of ␣2,6-sialylation of cell glycoproteins, as determined by reactivity with the Sambucus nigra lectin, is closely associated with the level of enzyme activity.