α-Latrotoxin (α-Ltx), a component of black widow spider venom, stimulates secretion from nerve terminals and from PC12 cells. In this study we examine the effects of expression of a newly cloned Ca2+-independent receptor for α-Ltx (CIRL) on secretion from bovine chromaffin cells. We first characterized the effect of α-Ltx on secretion from untransfected cells. α-Ltx, by binding in a Ca2+-independentmanner to an endogenous receptor, causes subsequent Ca2+-dependent secretion from intact cells. The stimulation of secretion is correlated with Ca2+influx caused by the toxin. In permeabilized cells in which the Ca2+concentration is regulated by buffer, α-Ltx also enhances Ca2+-dependent secretion, indicating a direct role of the endogenous receptor in the secretory pathway. Expression of CIRL increased the sensitivity of intact and permeabilized cells to the effects of α-Ltx, demonstrating that this protein is functional in coupling to secretion. Importantly, in the absence of α-Ltx, the expression of CIRL specifically inhibited the ATP-dependent component of secretion in permeabilized cells without affecting the ATP-independent secretion. This suggests that this receptor modulates the normal function of the regulated secretory pathway and that α-Ltx may act by reversing the inhibitory effects of the receptor.