To survive, neurons and other eukaryotic cells must rapidly repair (seal) plasmalemmal damage. Such repair occurs by an accumulation of intracellular vesicles at or near the plasmalemmal disruption. Diacylglycerol (DAG)-dependent and cAMP-dependent proteins are involved in many vesicle trafficking pathways. Although recent studies have implicated the signaling molecule cAMP in sealing, no study has investigated how DAG and DAG-dependent proteins affect sealing and, whether pharmacological inhibition of such proteins could promote immediate repair of damaged mammalian axons. To this end, I investigated the role of DAG, protein kinase C (PKC) and other DAG-activated proteins in plasmalemmal sealing in B104 neurons in vitro and rat sciatic nerves in vivo. Using dye exclusion to assess Ca2+-dependent vesicle-mediated sealing of transected neurites of individually identifiable rat hippocampal B104 cells, I now report that, compared to non-treated controls, sealing probabilities and rates are increased by DAG and cAMP analogs that activate PKC and Munc13-1, and protein kinase A (PKA). Sealing is decreased by inhibiting DAG-activated novel protein kinase C isozymes η (nPKCη) and θ (nPKCθ) and, Munc13-1, the PKC effector myristoylated alanine rich PKC substrate (MARCKS) or phospholipase C (PLC). DAG-increased sealing is prevented by inhibiting MARCKS or PKA. Sealing probability is further decreased by simultaneously inhibiting nPKCη, nPKCθ and PKA. Extracellular Ca2+, DAG or cAMP analogs do not affect this decrease in sealing. I also report that applying inhibitors of nPKC and PKA to rat sciatic axons crush-severed in vivo under physiological calcium, do not promote immediate repair by polyethylene glycol (PEG), as assessed by compound action potential conduction and dye diffusion through crush sites. These and other data suggest that DAG increases sealing through MARCKS and that nPKCη, nPKCθ and PKA are all required to seal plasmalemmal damage in B104 neurons, and likely all eukaryotic cells. / text