Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-alpha-D-galactosaminide results in profound changes in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosaccharide chains synthesized by HT-29 MTX cells and the changes induced by permanent drug exposure. Mucins from untreated cells contained nine monosialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4). Core 1 structures predominated, in particular NeuAcalpha2-3Galbeta1-3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-alpha-D-galactosaminide from days 2-21 resulted in a decrease in intracellular mucins and both their sialic acid and galactose content, and an increased T (Galbeta1-3GalNAcalpha-O-Ser/Thr) and Tn (GalNAcalpha-O-Ser/Thr) antigenicity. A 3-fold increase in both Galbeta1-3GalNAc alpha2, 3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-antigen was not detectable in mucin droplets in control cells, but was strongly expressed in intracytoplasmic vesicles in treated cells. In these cells, MUC1 and MUC3 transcripts were up-regulated, whereas MUC2, MUC5B and MUC5AC were down-regulated. Furthermore, constitutive and secretagogue-induced MUC5AC secretion was reduced and no mucus layer was detected. In conclusion, benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation and altered regulation of MUC5AC secretion.