Astroglial cells participate in a variety of developmental events during neuronal morphogenesis. We have shown that axonal, but not dendritic, outgrowth of spinal cord neurons can be promoted by a diffusible factor or factors secreted from target region-derived cerebellar astroglia in vitro in comparison with spinal astroglia. In the present study, we examined the involvement of protein kinase C (PKC) in the axon-promoting effect by astroglia. The inhibition of PKC by sphingosine or by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) at high concentration greatly reduced the mean axonal length of spinal neurons cultured in medium conditioned by cerebellar astroglia (SCn-CBg), while activation of PKC by TPA at low concentration, or by retinoic acid, was not additive to the glial effect. The activation of PKC by TPA or retinoic acid promoted axon growth of spinal neurons cultured in medium conditioned by spinal astroglia (SCn-SCg), which otherwise would not be as supportive for axon growth as cerebellar astroglia. Western blotting and PKC activity assays showed that there was a trend for increased PKC activity and protein levels (in particular, PKC beta) in SCn-CBg cultures, which correlated with enhanced axon growth. Inhibition of PKC by sphingosine appeared to decrease protein levels, especially PKC beta, which correlated with suppressed axon outgrowth. In SCn-SCg cultures, phorbol ester activation of PKC increased both activity and protein levels of both PKC alpha and PKC beta. This activation correlated with stimulated axonal outgrowth. These results suggest that the glial signaling that regulates specific axonal outgrowth by target astroglia is mediated in part by the PKC second messenger system.