The effects of dibutyryl cyclic AMP (B2cAMP) and theophylline on glyoprotein synthesis in rabbit colon were studied in mucosal organ cultures using [3H]glucosamine as a precursor. Addition of B2cAMP (1 mm) to culture medium caused a significant increase in glycoprotein synthesis after 12 and 24 hr compared with biopsies cultured in control medium. The increase in glycoprotein synthesis was observed only if the cyclic nucleotide was present continuously in the incubation medium for at least 12 hr. The stimulatory effect of B2cAMP on [3H]glucosamine incorporation was blocked by cycloheximide. B2cAMP also stimulated mucosal uptake of glucosamine into the intracellular pool and markedly enhanced specific activity of mucosa galactosyltransferase, an enzyme involved in glycoprotein synthesis. Addition of 5mM theophylline caused a greater than 2-fold increase in cAMP levels, which was also accompanied by an increase in glucosamine uptake and incorporation into mucosal glycoproteins. This study demonstrates that elevation of intracellular cAMP concentration in colon epithelium in vitro is associated with an increase in glycoprotein synthesis. These effects may be mediated in part by (1) increased uptake of glycoprotein precursors such as glucosamine, and (2) increased activity of glycoprotein synthetic enzymes.