cGMP-dependent protein kinase (cGMP kinase) has been implicated in the regulation of the cytosolic calcium level ([Ca2+]i). In Chinese hamster ovary (CHO) cells stably transfected with the cGMP kinase I alpha (CHO-cGK cells), cGMP kinase suppressed the thrombin-induced increase in inositol 1,4,5-trisphosphate and [Ca2+]i (Ruth, P., Wang, G.-X., Boekhoff, I., May, B., Pfeifer, A., Penner, R., Korth, M., Breer, H., and Hofmann, F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2623-2627). Cholecystokinin activated intracellular calcium release via a pertussis toxin (PTX)-insensitive pathway in CHO-cGK cells. cGMP kinase did not attenuate the CCK-stimulated [Ca2+]i. In contrast, cGMP kinase suppressed calcium influx stimulated by insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) via PTX-sensitive pathways. The effects of PTX and cGMP kinase on [Ca2+]i were not additive. 8-Bromo-cGMP had no effect on [Ca2+]i stimulated by IGF-1 or IGF-2 in wild type CHO cells. These results suggested that cGMP kinase inhibited the different signaling pathways by the phosphorylation of a PTX-sensitive G protein. cGMP kinase phosphorylated the alpha subunits of Gi1, Gi2, and Gi3 in vitro. Phosphorylation stoichiometry was 0.4 mol of phosphate/mol of G alpha i1 after reconstitution of heterotrimeric Gi1 in phospholipid vesicles. The alpha subunit of Gi was also phosphorylated in vivo. These results show that cGMP kinase blocks transduction of distinct hormone pathways that signal via PTX-sensitive Gi proteins.