Renal epithelial cell H+ secretion is an exocytic-endocytic phenomenon. In the inner medullary collecting duct (IMCD) cell line, which we have utilized as a model of renal epithelial cell acid secretion, we found previously that acidification increased exocytosis and alkalinization increased endocytosis. It is likely, therefore, that the rate of proton secretion is regulated by the membrane insertion and retrieval of proton pumps. There is abundant evidence from studies in the nerve terminal and the chromaffin cell that vesicle docking, membrane fusion, and discharge of vesicular contents (exocytosis) involve a series of interactions among so-called trafficking proteins. The clostridial toxins, botulinum and tetanus are proteases that specifically inactivate some of these proteins. In these experiments we demonstrated, by immunoblot and immunoprecipitation, the presence in this IMCD cell line of the specific protein targets of these toxins, synaptobrevin/vesicle-associated membrane proteins (VAMP), syntaxin, and synaptosomal-associated protein-25 (SNAP-25). Furthermore, we showed that these toxins markedly inhibit the capacity of these cells to realkalinize after an acid load. Thus these data provide new insight into the mechanism for H+ secretion in the IMCD.