Epithelial cells of the colonic crypt engage in cAMP-mediated fluid and electrolyte secretion. In addition to participating in electrolyte transport, colonic crypt cells also synthesize and secrete a number of proteins and peptides that play a crucial role in mucosal homeostasis. In the present study we show that cAMP regulates not only electrolyte secretion but also polarized protein secretion in a tissue culture model of colonic crypt cells. We found that apical but not basolateral protein secretion was stimulated by a physiological activator of the cAMP pathway, vasoactive intestinal peptide, as well as by a cell-permeant analogue of cAMP (8-(4-chlorophenylthio)cAMP) at concentrations as low as 12.5 μM. Based on several criteria, we determined that the regulation of protein secretion by cAMP in HT29-CL19A cells occurs via stimulation of constitutive membrane traffic from the trans- Golgi network (TGN) to the apical cell surface. In addition, the regulation of apical protein secretion by cAMP was Cl --dependent with cAMP inhibiting rather than stimulating secretion in Cl --depleted cells. The locus of cAMP action on the secretory pathway is at least in part at the level of the TGN, where it stimulates the sialylation of α1-antitrypsin (i.e. one of the identified secretory proteins) in addition to the traffic of secretory proteins from the TGN to the apical cell surface. We propose that a cyclic AMP and Cl --dependent regulation of TGN acidification could modulate both sialylation and secretory vesicle budding at the TGN.