Rat brain synaptosomes were isolated to study the effects of protein kinase inhibitors (sphingosine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide, staurosporine) on Ca²⁺-dependent and Ca²⁺-independent [¹⁴C]GABA release. The Ca²⁺-dependent [¹⁴C]GABA release was stimulated by depolarization with a K⁺-channel blocker, 4-aminopyridine, or high K⁺ concentration. It has been shown that 4-aminopyridine-evoked [¹⁴C]GABA release strongly depends on extracellular Ca²⁺ while K⁺-evoked [¹⁴C]GABA release only partly decreases in the absence of calcium. The substitution of sodium by choline in Ca²⁺-free medium completely abolished Ca²⁺-independent part of K⁺-evoked [¹⁴C]GABA release. So the main effect of 4-aminopyridine is the Ca²⁺-dependent one while high K⁺ is able to evoke [¹⁴C]GABA release in both a Ca²⁺-dependent and Na⁺-dependent manner. In experiments with protein kinase inhibitors, 4-aminopyridine and high K⁺ concentration were used to study the Ca²⁺-dependent and the Ca²⁺-independent [¹⁴C]GABA release, respectively. In addition, the Ca²⁺-independent [¹⁴C]GABA release. was studied using α-latrotoxin as a tool. Pretreatment of synaptosomes with protein kinase inhibitors tested, except of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, resulted in a marked inhibition of 4-aminopyridine-stimulated Ca²⁺-dependent [¹⁴C]GABA release. The inhibitory effects of N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide and staurosporine on [¹⁴C]GABA release were not due to their effects on 4-aminopyridine-promoted ⁴⁵Ca²⁺ influx into synaptosomes. Only sphingosine (100μM) reduced the ⁴⁵Ca²⁺ influx. All the inhibitors investigated were absolutely ineffective in blocking the Ca²⁺-independent [¹⁴C]GABA release stimulated by α-latrotoxin. Three of them, except for sphingosine, did not affect the Ca²⁺-independent [¹⁴C]GABA release stimulated by high potassium. The inhibitory effect of sphingosine was equal to 30%. Thus, if [¹⁴C]GABA release occurred in a Ca²⁺-independent manner irrespective of whether α-latrotoxin or high K⁺ stimulated this process, it was not inhibited by the drugs decreased the Ca²⁺-dependent [¹⁴C] GABA release. Given the above points it is therefore not unreasonable to assume that the absence of Ca²⁺ in the extracellular medium created the conditions in which the activation of neurotransmitter release was not accompanied by Ca²⁺-dependent dephosphorylation of neuronal phosphoproteins, and as a consequence the regulation of exocytotic process was modulated so that the inhibition of protein kinases did not disturb the exocytosis.