Rat gastric mucosa segments incorporate [3H]glucosamine and [35S]sulfate into glycosaminoglycans and glycoproteins. After 6-h incubation, the order of radioactivity incorporated from both precursors was glycoproteins greater than heparan sulfate greater than chondroitin sulfate. Pulse-chase studies indicate that much of the tissue-bound radioactivity was extruded into the medium by the 6th hour of incubation. The glycoprotein fraction was electrophoretically polydisperse and its subfractions exhibited a wide range of 3H/35S. Mild acid hydrolysis of the glycoprotein fraction afforded sulfated hexosamine fractions which migrated like N-acetylhexosamine mono- and disulfates by paper chromatography and by paper electrophoresis and exhibited 3H/35S which was in accord with the presence of mono- and disulfated compounds. The ratio of the monosulfated to the disulfated species was about 7 to 1. In separate studies, both of these compounds were found in the glycoprotein fraction elaborated into the medium, the glycoprotein from the combined tissue plus medium, and in the sulfated oligosaccharide isolated from the latter by reductive alkaline cleavage. Subsequent acid hydrolysis and paper chromatographic examination of the sugars liberated from each of these compounds revealed the presence of glucosamine as the principal amino sugar. The results of a periodate oxidation study of the N-acetylhexosamine monosulfate were in accord with N-acetylhexosamine 6-sulfate being the principal compound. About 20% of the 3H in the N-acetylhexosamine monosulfate and 10% of the 3H in the disulfated compound were found associated with galactosamine. Separate incorporation studies with [35S] sulfate and [3H]glucose not only confirmed the presence of N-acetylhexosamine mono- and disulfates in the glycoprotein fraction but also demonstrated the presence of galactose monosulfate therein.