The regulation of renin release is unusual in that intracellular calcium reportedly acts as an inhibitory second messenger. Increased calcium not only inhibits renin release but is a cofactor in nitric oxide synthesis. Also, nitric oxide can inhibit renin release. This study was done in vitro using rat renal cortical slices to determine whether calcium-mediated renin inhibition could be in part due to the concurrent production of nitric oxide. Renin concentration in the incubation medium was determined by radioimmunoassay for angiotensin I (Ang I) generation (in nanograms Ang I per hour per milligram per 30 minutes of incubation). In all studies, n=6 to 17. In normal-calcium incubation medium (2.6 mmol/L), 10−4 mol/L NG-monomethyl L-arginine, which blocks nitric oxide synthesis, caused an 18% increase in basal renin release (78.6±8.9 versus 66.7±5.8 [ng Ang I/h]/mg per 30 minutes incubation, P<.05). When calcium was eliminated from the incubation medium, basal renin release doubled (to 133.1±15.2 [ng Ang I/h]/mg per 30 minutes incubation, P<.001). Without calcium, inhibiting nitric oxide synthesis had no further effect on renin release (126.8±17.7 [ng Ang I/h]/mg per 30 minutes incubation). High-calcium medium (7.8 mmol/L) reduced basal renin release by half (30.8±4.8 [ng Ang I/h]/mg per 30 minutes incubation, P<.001), but inhibiting nitric oxide synthesis in high-calcium medium stimulated renin release by 50% (46.9±6.2 [ng Ang I/h]/mg per 30 minutes incubation, P<.005). Addition of the calmodulin inhibitor W-7 completely reversed the inhibition of renin by high-calcium medium. Incubation of cortical slices with angiotensin II also resulted in a 45% suppression of basal renin release (P<.005), and this effect could be blocked by either removal of calcium from the incubation medium or inhibition of nitric oxide synthesis. Thus, nitric oxide synthesis inhibition increased basal renin release in proportion to the amount of calcium in the incubation medium. Furthermore, angiotensin inhibited renin by a calcium- and apparently a nitric oxidedependent pathway. Because nitric oxide can inhibit renin release, these results suggest that at least part of the inhibitory influence of calcium on renin release can be attributed to calcium-dependent nitric oxide synthesis.