We have recently shown that several putative selective inhibitors of Ca²⁺-calmodulin-dependent myosin light chain kinase (MLCK), such as ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], reversibly stimulate renin secretion [C. S. Park, S.-H. Chang, H. S. Lee, S.-H. Kim, J. W. Chang, and C. D. Hong. Am. J. Physiol. 271 (Cell Physiol. 40): C242-C247, 1996]. We hypothesized that Ca²⁺ inhibits renin secretion, via phosphorylation of 20-kDa myosin light chain (MLC₂₀), by activating MLCK. In the present studies, we have investigated the types of protein phosphatase (PP) involved in the control of renin secretion through inhibition of MLC dephosphorylation using inhibitors of various types of serine/threonine-specific protein phosphatases. Cyclosporin A, a putative inhibitor of PP type 2 (calcineurin), was without effect. Calyculin A and okadaic acid, putative selective inhibitors of both PP type 1 (PP1) and type 2A (PP2A), significantly inhibited renin secretion under control conditions. Calyculin A had inhibitory effects at least 10-fold more potent than okadaic acid, suggesting that PP1, rather than PP2A, is involved in the control of renin secretion. Furthermore, calyculin A blocked the reversal of renin secretion preinhibited by raised intracellular Ca²⁺ concentrations in a concentration-dependent manner. Calyculin A (10⁶ M) significantly inhibited renin secretion stimulated by lowering intracellular Ca²⁺ concentrations and blocked the stimulatory effect of ML-9 on renin secretion. Taking all of these results into consideration, we hypothesize that dephosphorylation of MLC₂₀ by Ca²⁺-independent PP1 stimulates renin secretion, whereas phosphorylation of MLC₂₀ by Ca²⁺-calmodulin-dependent MLCK inhibits it. This hypothesized regulatory model of renin secretion predicts that the rate of renin secretion at a given time is determined by the ratio of phosphorylated to dephosphorylated MLC₂₀, which is, in turn, determined by the dynamic balance between activity of MLCK and MLC phosphatase.