The proliferation of cerebral endothelial cells is a crucial step in neural angiogenesis and is a process responsive to changes in the surrounding environment. Serum-free medium conditioned by rat cortical astrocytes was found to accelerate DNA synthesis, induce transient activation of protein kinase C (PKC), and increase the endogenous phosphorylation of the PKC-specific substrate, the 85 kDa MARCKS protein, in rat cerebromicrovascular endothelial cells (RCEC). The stimulatory factor(s) in astrocyte conditioned media (ACM) were heat- and trypsin-sensitive and found to have an apparent molecular weight greater than 10 kDa. The potent PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), also stimulated RCEC proliferation, whereas the inhibition of PKC by staurosporine caused a concomitant loss in ACM-induced PKC translocation, MARCKS protein phosphorylation and DNA synthesis. These findings implicate PKC activation as a critical early event in cerebral endothelial cell proliferation triggered by astrocyte-derived mitogen(s).