Luminal Ca2+ has been proposed to regulate the sensitivity of intracellular Ca2+ stores to InsP3 and perhaps thereby to contribute to the mechanisms responsible for regenerative intracellular Ca2+ signals. Since Ca2+ release from intracellular stores is accompanied by K+ influx into the stores, InsP3-stimulated Ca2+ mobilization can be inhibited by substantially reducing the [K+] of incubation media. By measuring [3H]InsP3 binding to permeabilized hepatocytes in K(+)-deficient media, thereby preventing InsP3-stimulated Ca2+ mobilization, we have examined the effects of Ca2+ within the intracellular stores on equilibrium binding of InsP3 to its receptor. Our results demonstrate a small, but significant (P < 0.05, n = 8-9), effect of luminal Ca2+ on InsP3 binding; the concentration of InsP3 causing half-maximal displacement of [3H]InsP3 (1.25 nM) was 3.5 nM for empty stores and 2.1 nM for stores containing Ca2+. Our results suggest that at least part of the stimulatory effect of luminal Ca2+ on InsP3-stimulated Ca2+ mobilization may result from an effect of luminal Ca2+ on InsP3 binding to its receptor.