Protein kinase A and protein kinase C are involved in processes that enhance glutamate release at glutamatergic nerve terminals. However, it is not known whether these two kinases co-exist within the same nerve terminal, nor is it clear what impact their simultaneous activation may have on neurotransmitter release. In cerebrocortical nerve terminals, co-application of forskolin, which increases cAMP levels and activates protein kinase A, and 4beta-phorbol dibutyrate, a direct activator of protein kinase C, synergistically enhanced the spontaneous release of glutamate. This enhancement exhibited both tetrodotoxin-sensitive and tetrodotoxin-resistant components. Interestingly, the tetrodotoxin-resistant component of release was not observed when cyclic AMP-dependent protein kinase (PKA) and calcium- and phospholipid-dependent protein kinase (PKC) were activated separately, but developed slowly after the co-activation of the two kinases, accounting for 50% of the facilitated release. This release component was dependent on voltage-dependent Ca2+ channels that opened spontaneously after PKA and PKC activation and occurred in the absence of Na+ channel firing. These data provide functional evidence for the co-existence of PKA- and PKC-signalling pathways in a subpopulation of glutamatergic nerve terminals.