Abstract. Individual isoforms of the protein kinase C (PKC) family of kinases may have assumed distinct responsibilities for the control of complex and diverse cellular functions. In this study, we show that an isoform specific interaction between PKCe and filamentous actin may serve as a necessary prelude to the enhancement of glutamate exocytosis from nerve terminals. Using a combination of cosedimentation, overlay, and direct binding assays, we demonstrate that filamentous actin is a principal anchoring protein for PKCe within intact nerve endings. The unusual stability and direct nature of this physical interaction indicate that actin filaments represent a new class of PKC-binding protein. The binding of PKCe to actin required that the kinase be activated, presumably to expose a cryptic binding site that we have identified and shown to be located between the first and second cysteine-rich regions within the regulatory domain of only this individual isoform of PKC. Arachidonic acid (AA) synergistically interacted with diacylglycerol to stimulate actin binding to PKCe. Once established, this protein-protein interaction securely anchored PKCe to the cytoskeletal matrix while also serving as a chaperone that maintained the kinase in a catalytically active conformation. Thus, actin appears to be a bifunctional anchoring protein that is specific for the PKCe isoform. The assembly of this isoform-specific signaling complex appears to play a primary role in the PKC-dependent facilitation of glutamate exocytosis.VOLUTION has conserved a fundamental mechanism that ensures the sorting and fusion of secretory vesicles upon their delivery to appropriate destinations along the secretory pathway. In many eukaryotes, however, alternative pathways have emerged in which vesicle delivery and fusion have become rigorously regulated events that are capable of manifesting use-dependent variations in the probability of their occurrence. Glutamate exocytosis is a particularly striking example of a secretory event that, in presynaptic nerve terminals, has acquired a highly evolved mechanism for regulating the efficiency of excitatory synaptic transmission. Moreover, it has been established that use-dependent changes in the reliability (Stevens and Wang, 1994) and extent (Schulz et al., 1994) of glutamate exocytosis directly participate in certain forms of synaptic plasticity.Studies from many laboratories suggest that a conditional relationship may exist between the efficiency of glutamate release and the activity of presynaptic protein