Calmodulin inhibits both inositol 1,4,5-trisphosphate (IP₃) binding to, and IP₃-evoked Ca²⁺ release by, cerebellar IP₃ receptors [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627–11632]. In the present study, full-length rat type-1 and -3 IP₃ receptors were expressed at high levels in insect Spodoptera frugiperda 9 cells and the effects of calmodulin were examined. In the absence of Ca²⁺, calmodulin caused a concentration-dependent and reversible inhibition of [³H]IP₃ binding to type-1 IP₃ receptors by decreasing their apparent affinity for IP₃. The effect was not reproduced by high concentrations of troponin C, parvalbumin or S-100. Increasing the medium free [Ca²⁺] ([Ca²⁺]_m) inhibited [³H]IP₃ binding to type-1 receptors, but the further inhibition caused by a submaximal concentration of calmodulin was similar at each [Ca²⁺]_m. In the absence of Ca²⁺, ¹²⁵I-calmodulin bound to a single site on each type-1 receptor subunit and to an additional site in the presence of Ca²⁺. There was no detectable binding of ¹²⁵I-calmodulin to type-3 receptors and binding of [³H]IP₃ was insensitive to calmodulin at all [Ca²⁺]_m. Both peptide and conventional Ca²⁺–calmodulin antagonists affected neither [³H]IP₃ binding directly nor the inhibitory effect of calmodulin in the absence of Ca²⁺, but each caused a [Ca²⁺]_m-dependent reversal of the inhibition of [³H]IP₃ binding caused by calmodulin. Camstatin, a peptide that binds to calmodulin equally well in the presence or absence of Ca²⁺, reversed the inhibitory effects of calmodulin on [³H]IP₃ binding at all [Ca²⁺]_m. We conclude that calmodulin specifically inhibits [³H]IP₃ binding to type-1 IP₃ receptors: the first example of a protein regulated by calmodulin in an entirely Ca²⁺-independent manner. Inhibition of type-1 IP₃ receptors by calmodulin may dynamically regulate their sensitivity to IP₃ in response to the changes in cytosolic free calmodulin concentration thought to accompany stimulation of neurones.