Cultured rat cerebellar granule cells depolarized by high KCl, display a large component of Ca2+ influx through L-type voltage-dependent Ca2+ channels as defined by a sensitivity to 1 μm nifedipine. This Ca2+ influx is not coupled to neurotransmitter exocytosis but has implications for neuronal development. KCl stimulation in the absence of external Ca2+ followed by the re-addition of Ca2+ allows the coupling of a class of L-type Ca2+ channels to neurotransmitter exocytosis as assessed by loading of glutamatergic pools with [3H]-d-aspartate. KCl stimulation in the absence of external Ca2+ (‘predepolarization’) enhances tyrosine phosphorylation of several cellular proteins, and inhibitors of tyrosine kinases block both phosphorylation and the neurotransmitter release coupled to the L-type Ca2+ channel. More specifically, an inhibitor of src family tyrosine kinases, PP1, blocks the effects of predepolarization suggesting a role for a src family kinase in the process. Furthermore, L-type Ca2+ channel recruitment and modulation of release could be activated with the tyrosine phosphatase inhibitor sodium orthovanadate. The phosphoproteins enhanced by predepolarization, which include the cytoskeletal proteins focal adhesion kinase (FAK) and vinculin, are also highly phosphorylated early on in culture when neurite outgrowth occurs. As the neurons develop a network of neurites, both tyrosine phosphorylation and L-type Ca2+ channel activity decrease. These results show a novel mechanism for the recruitment of L-type Ca2+ channels and their coupling to neurotransmitter release which involves tyrosine phosphorylation. This phenomenon has a role in cerebellar granule cell development.