Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells^,. Calcium release from inositol 1,4,5-trisphosphate (IP₃)-sensitive stores is negatively regulated by binding of calmodulin to the IP₃ receptor (IP₃R)^, and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway^,. Activation of cGKI decreases IP₃-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIβ phosphorylated the IP₃R and cGKIβ, and a protein of relative molecular mass 125,000 which we now identify as the IP₃R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP₃R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIβ, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIβ in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP₃-induced calcium release.