In order to gain insights into the regulatory mechanisms of epithelial protein secretion we analyzed the secretion of metabolically labeled proteins by polarized epithelial cells. We found that cAMP stimulates apical but not basolateral protein secretion in HT29-CL19A colonie epithelial cells by facilitating constitutive membrane traffic from the trans Golgi network (TGN) to the apical cell surface. This regulation of apical constitutive protein secretion was dependent on the presence of Cl", when Cl was replaced in the media with gluconate (i.e., an anion that is impermeable for most CI" channels), the regulation of protein secretion by cAMP was abolished. Cyclic AMP also stimulated the rate by which α1-antitrypsin, one of the dominant secretory proteins in HT29-CL19A cells, was sialylated in the TGN. Furthermore, our recent data indicate that the heterologous expression of WTCFTR in LLCPK (porcine kidney epithelial cells) introduces a regulation of apical protein secretion by cAMP that was not observed in untransduced or AFSOSCFTR-expressing LLCPK cells. These data support the notion that CFTR regulates apical protein secretion in polarized epithelial cells in a cAMP-dependent manner, in agreement with earlier reports implicating CFTR Cl- conductance in TGN acidification, protein sialylation and membrane traffic regulation in epithelial cells. Acknowledgment: LLCPK cells stably transduced by WT and AF508CFTR were kindly provided for these studies by Dr Seng Cheng (Genzyme Corporation).