The effect of KN62 (1-[N,O-bis(5-isoquinolinesulphonyl)-N -methyl-L-tyrosyl]-4-phenylpiperazine), a putative inhibitor of Ca/calmodulin-dependent kinase II (Ca/CaM-K II), on glutamate release from isolated nerve-terminals (synaptosomes) was examined. The drug caused a potent inhibition of KCl- and 4-aminopyridine-evoked glutamate release from isolated nerve-terminals (synaptosomes). Examination of the effect of the inhibitor on Ca(2+)-influx revealed that the diminution of glutamate release could be attributed to a decrease in cytosolic Ca. A direct effect of KN62 on synaptosomal Ca(2+)-channels was confirmed in experiments where Ba, which does not support CaM-dependent processes, was used in place of Ca. Additionally, whole-cell patch-clamping of cerebellar granule neurones directly demonstrated inhibition of Ca-currents by KN62. We therefore suggest that, in cellular systems, conclusions based on the use of KN62 as a Ca/CaM-K II blocker may be ambiguous and should be viewed with caution unless the effect of the drug on Ca-influx has also been quantified. The effect of KN62 on Ca(2+)-influx appears to be specific to slowly-or non-inactivating conductances, and therefore presents KN62 as a potentially useful tool in this context.