Using sheep cerebellum microsomes previously loaded with 45Ca2+ or 90Sr2+, we measured the dependence of inositol 1,4,5-trisphosphate (InsP3)-induced efflux of these ions on Ca2+ or Sr2+ on the cytosolic side. At a low InsP3 concentration, Ca2+ in the submicromolar range only poorly activated 45Ca2+ or 90Sr2+ efflux, and higher Ca2+ concentrations were inhibitory. In contrast, Sr2+ in the micromolar range activated release efficiently, while only very high Sr2+ concentrations were inhibitory. Experiments were repeated in the presence of a high InsP3 concentration, which allowed increasing free Ca2+ to micromolar concentrations without inducing complete inhibition of the InsP3-dependent efflux. Under these conditions, micromolar Ca2+ was found to activate efflux to a large extent, similar to that previously found with Sr2+. Optimal activation by Ca2+ of the InsP3-dependent channel occurs at micromolar rather than submicromolar free Ca2+ concentrations, but at too low an InsP3 concentration, Ca(2+)-induced activation is counteracted by Ca(2+)-induced inactivation. Separate measurements of [3H]-InsP3 binding at a low concentration showed that Sr2+ and Ca2+ did not enhance the amount of bound [3H]-InsP3, implying that the activating effect of Sr2+ and Ca2+ in cerebellar microsomes is mediated by an increase in the channel opening probability and not by an increase in the receptor's affinity for InsP3. A similar relationship also holds in the case of the activating effect of nucleotides.