Gadolinium chloride is a potent blocker of voltage-sensitive Ca2+ channels. When added to fura-2 loaded rat cortical synaptosomes 1 min before depolarization with 33 mM KCl, it causes a dose-dependent inhibition of the resulting rise in intrasynaptosomal free Ca2+ with an IC50 of approximately 10 microM. The effect of GdCl3 on intrasynaptosomal free Ca2+ is not accompanied by an equivalent effect on Ca(2+)-dependent glutamate release. In the presence of 100 microM GdCl3, 33 mM KCl does not produce a detectable change in the fura-2 signal but Ca(2+)-dependent glutamate release is only reduced by around 12%. Using BaCl2, which is less effectively buffered in synaptosomes than Ca2+, we have shown that there is residual KCl-stimulated divalent cation entry into synaptosomes in the presence of 100 microM GdCl3. These data, combined with those from other laboratories, strengthen the argument for localized Ca2+ entry through Ca2+ channels linked to neurotransmitter release from synaptosomes and add to the evidence that the channels may exhibit considerable neurotransmitter specificity.