In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of cysteine string protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis. Prior work indicated that csp is associated with cortical granules of Xenopus oocytes. In oocytes exhibiting >20-fold overexpression of full-length Xenopus csp, cortical granule exocytosis was reduced by ϳ80%. However, csp overexpression did not affect constitutive exocytosis. Subcellular fractionation and confocal fluorescence microscopy revealed that little or none of the overexpressed csp was associated with cortical granules. This accumulation of csp at sites other than cortical granules suggested that mislocalized csp might sequester a protein that is important for regulated exocytosis. Because the NH 2 -terminal region of csp includes a J-domain, which interacts with constitutively expressed 70-kDa heat shock proteins (Hsc 70), we evaluated the effect of overexpressing the J-domain of csp. Although the native J-domain of csp inhibited cortical granule exocytosis, point mutations that interfere with J-domain binding to Hsc 70 eliminated this inhibition. These data indicate that csp interaction with Hsc 70 molecular chaperones is vital for regulated secretion in Xenopus oocytes.Eggs of many species exhibit cortical granule exocytosis, an early postfertilization event that leads to a sustained block of polyspermy (1). Although oocytes of Xenopus laevis must undergo maturation before sperm can elicit cortical granule exocytosis, immature oocytes (and eggs) of Xenopus secrete in response to activators of protein kinase C (2). Somewhat unexpectedly (given that the majority of regulated secretory events are initiated by an increase of cytosolic Ca 2ϩ , Refs. 3 and 4), cortical granule exocytosis in Xenopus oocytes is insensitive to changes of cytosolic Ca 2ϩ . For instance, this secretory event is not triggered by Ca 2ϩ ionophores (using physiological concentrations of Ca 2ϩ ); it cannot be evoked by direct injection of Ca 2ϩ into the cytoplasm, and it is unaffected by removal of extracellular Ca 2ϩ or by buffers that clamp cytosolic Ca 2ϩ below the resting level (5-7). Thus, oocytes offer an empirical avenue for assessing the function of proteins in a secretory pathway where calcium ions play no direct role. This issue is of considerable importance with respect to cysteine string proteins (csp 2 (s)), a class of proteins found associated with a broad range of regulated secretory organelles (8 -10).Csp was originally identified as a novel synaptic antigen in Drosophila (11), and several subsequent investigations indicated that csp might be important as a modulator of presynaptic Ca 2ϩ channels (12-17). However, it also became evident that regulation of presynaptic Ca 2ϩ channe...