To examine the role of the calcium/calmodulin-dependent phosphatase calcineurin in regulation of renin release, we assayed exocytosis using whole-cell patch clamp of single juxtaglomerular cells in culture. The calcineurin inhibitor, cyclosporine A (CsA), significantly increased juxtaglomerular cell membrane capacitance, an index of cell surface area and an established measure of exocytosis in single-cell assays. This effect was mimicked by intracellular delivery of a calcineurin inhibitory peptide, the calcium chelator ethylene glycol tetraacetic acid (EGTA), or the calmodulin inhibitor W-13. Simultaneous exposure to EGTA and CsA had no additive effect. The protein kinase A (PKA) blocker RpcAMPs had no effect on the CsA-induced increase in membrane capacitance. Intra- and extracellular application of tacrolimus did not alter membrane capacitance. A calmodulin antagonist (calmidazolium) and CsA, but not tacrolimus, significantly stimulated renin release from cultured juxtaglomerular cells. Juxtaglomerular cells expressed the calcineurin isoforms A-beta and A-gamma but not A-alpha. Plasma renin concentrations (PRCs) were not different in wild-type, calcineurin A-alpha, or A-beta knockout mice but increased after CsA treatment of the A-alpha knockout, while renin mRNA was suppressed. We conclude that calcineurin and calcium/calmodulin suppress exocytosis of renin from juxtaglomerular cells independent of PKA.