The heat-stable enterotoxin of Escherichia coli (STa) is a potent stimulant of intestinal chloride and bicarbonate secretion. Guanylyl cyclase C (GC-C) has been shown to be the primary receptor involved in mediating this response. However, numerous studies have suggested the existence of an alternative STa-binding receptor. The aims of this study were to determine whether a non-GC-C receptor exists for STa and what is the functional relevance of this for intestinal bicarbonate secretion in mice. ¹²⁵I-STa-binding experiments were performed with intestinal mucosae from GC-C knockout (KO) and wild type (WT) mice. Subsequently, the functional relevance of an alternative STa-binding receptor was explored by examining STa-, uroguanylin-, and guanylin-stimulated duodenal bicarbonate secretion (DBS) in GC-C KO mice in vitro and in vivo. Significant ¹²⁵I-STa-binding occurred in the proximal small intestines of GC-C KO and WT mice. Analysis of binding coefficients and pH dependence showed that ¹²⁵I-STa-binding in GC-C KO mice involved a receptor distinct from that of WT mice. Functionally, STa, uroguanylin, and guanylin all stimulated a significant increase in DBS in GC-C KO mice. Uroguanylin- and guanylin-stimulated DBS were significantly inhibited by glibenclamide, but not by 4,4`-diisothiocyanato-stilbene-2,2`-disulfonic acid (DIDS). However, STa-stimulated DBS was unaffected by glibenclamide but inhibited by DIDS. Taken together, our results suggest that alternative, non-GC-C, receptors likely exist for STa, uroguanylin, and guanylin in the intestines of mice. While uroguanylin- and guanylin-stimulated DBS are cystic fibrosis transmembrane conductance regulator (CFTR) -dependent, STa-stimulated DBS is CFTR independent. Further understanding of this alternative receptor and its signaling pathway may provide important insights into rectification of intestinal bicarbonate secretion in cystic fibrosis.—Sellers, Z. M., Mann, E., Smith, A., Ko, K. H., Giannella, R., Cohen, M. B., Barrett, K. E., Dong, H. Heat-stable enterotoxin of Escherichia coli (STa) can stimulate duodenal HCO₃^- secretion via a novel GC-C- and CFTR-independent pathway.