The increase in cytosolic free Ca2+ concentration that follows mobilization of intracellular Ca2+ stores by inositol 1,4,5-trisphosphate Ins(1,4,5)P3 has been reported to modulate the sensitivity of Ins(1,4,5)P3 receptors. We have examined the effects of cytosolic Ca2+ on Ins(1,4,5)P3-stimulated Ca2+ mobilization in permeabilized hepatocytes. Increasing the free Ca2+ concentration in the medium ([Ca2+]m) caused a concentration-dependent increase in the sensitivity of the stores to Ins(1,4,5)P3; the concentration of Ins(1,4,5)P3 that caused half-maximal Ca2+ mobilization (EC50) decreased from 261 +/- 11 nM (n = 3) to 50 +/- 4 nM (n = 8) as [Ca2+]m was increased from approximately 7 nM to 1.6 microM. The EC50 for this effect of Ca2+ was approximately 250 nM. In addition, higher [Ca2+]m (> 600 nM) reduced the extent of Ca2+ release induced by a maximal concentration of Ins(1,4,5)P3; elevating [Ca2+]m to 2.6 microM reduced the proportion of Ca2+ releasable by Ins(1,4,5)P3 by 94 +/- 8% (n = 3). Both effects of Ca2+ were independent of Ca2(+)-stimulated Ins(1,4,5)P3 formation. When elevated [Ca2+]m was returned to control levels, the sensitization of Ins(1,4,5)P3-mediated Ca2+ mobilization reversed completely, whereas the reduction in the size of the Ins(1,4,5)P3-sensitive Ca2+ pool was reversed by only 59 +/- 12% (n = 5) after 20 s and was not further reversed after 100 s. The two distinct effects of Ca2+ on Ins(1,4,5)P3-mediated Ca2+ release combined to control the amount of Ca2+ released by a submaximal concentration of Ins(1,4,5)P3 in a biphasic manner.