Little is known about the effects of nitric oxide (NO) and the cyclic GMP (cGMP)/protein kinase G (PKG) system on Ca²⁺ signaling in vascular smooth muscle cells (VSMC) of resistance vessels in general and afferent arterioles in particular. We test the hypotheses that cGMP-, Ca²⁺ dependent big potassium channels (BK_Ca2+) buffer the Ca²⁺ response to depolarization by high extracellular KCl and that NO inhibits adenosine diphosphoribose (ADPR) cyclase, thereby reducing the Ca²⁺-induced Ca²⁺ response (CICR). We isolated rat afferent arterioles utilizing the magnetized microsphere method and measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) with fura-2, a preparation in which endothelial cells do not participate in [Ca²⁺]_i responses. KCl (50 mM) - induced depolarization causes an immediate increase in [Ca²⁺]_i of 151 nM. The blockers L-NAME (of nitric oxide synthase), ODQ (of guanylyl cyclase), KT5823 (of PKG activation) or iberiotoxin (IBX, of BK_Ca2+ activity) do not alter the [Ca²⁺]_i response to KCl, suggesting no discernible endogenous NO production under basal conditions. The NO donor sodium nitroprusside (SNP) reduces the [Ca²⁺]_i response to 77 nM; IBX restores the response to control values. These data show that activation of BK_Ca2+ in the presence of NO/cGMP provides a brake on KCl - induced [Ca²⁺]_i responses. Experiments with the inhibitor of cyclic ADPR, 8-Br cADPR and SNP + downstream inhibitors of PKG and BK_Ca2+ suggest that NO inhibits ADPR cyclase in intact arterioles. When we pretreat afferent arterioles with 8-Br cGMP (10μ M), the response to KCl is 143 nM. However, in the presence of both IBX and 8-Br cGMP, we observe a surprising doubling of the [Ca²⁺]_i response to KCl. In summary, we present evidence for effects of the NO/cGMP/PKG system to reduce [Ca2+]i, via activation of BK_Ca2+ and possibly by inhibition of ADPR cyclase and to increase [Ca²⁺]_i, by a mechanism(s) yet to be defined.