The mechanisms of proguanylin synthesis and secretion in the intestine are incompletely understood. We designed an in vitro model to study proguanylin secretion in a single, homogenous population of intestinal villous epithelial cells. C2Bbe1 cells, a differentiated sub-clone of Caco-2 cells, were used to examine the direction of proguanylin secretion and the potential for feedback regulation via activators of the guanylyl cyclase C (GC-C)signal transduction pathway. When cells were grown on transwell inserts, proguanylin was secreted into the apical and basolateral media, consistent with other models of intestinal guanylin secretion. Proguanylin synthesis and secretion was not decreased upon activation of GC-C mediated chloride secretion, implying a regulatory system other than simple feedback inhibition. These data describe the use of C2Bbe1 cells as a model for proguanylin secretion in villous epithelial cells and demonstrate their potential use for the study of the regulatory mechanisms governing proguanylin sysnthesis and secretion.