Inositol trisphosphate (InsP3) mediated Ca2+ release is modulated in a complex way by Ca2+. It is not known how the InsP3 receptor responds to a slowly increasing cytosolic [Ca2+]. Two different cell lines (A7r5 smooth-muscle cells and EBTr cells from tracheal mucosa) were investigated. We have now stimulated the Ca2+ stores of the permeabilized cells with a near-threshold [InsP3] and then increased the cytosolic [Ca2+] from 5 nM to 3 microM in 55 steps each lasting 6 s. The rate of InsP3-induced Ca2+ release abruptly increased around 100 nM Ca2+ and reached a maximum at 300 nM Ca2+, above which the Ca2+ release became smaller. The stimulatory effect of cytosolic Ca2+ was much less than that induced by elevating the [InsP3]. The time course of activation by Ca2+ in permeabilized cells resembles the fast InsP3-induced Ca2+ release following the pacemaker [Ca2+] rise in the agonist-stimulated intact cell.