Mucin desulfation is believed to be a rate-limiting step in mucin degradation by colon bacteria. The activities of enzymes hydrolysing nine linkages found in mucin oligosaccharide chains were measured using model substrates, in extracts of two mucin-degrading bacteria, Prevotella strain RS2 and Bacteroides fragilis. Sulfatases desulfating N-acetylglucosamine-6-sulfate, galactose-6-sulfate and galactose-3-sulfate were found. The genomic DNA downstream from the gene encoding the mucin-desulfating sulfatase (N-acetylglucosamine-6-sulfatase) in Prevotella was sequenced, and two putative genes identified which are likely to be coexpressed with this sulfatase, though their activities are unknown. Northern and Western analyses showed that expression of this short operon of three genes is increased during growth on mucin.