Two-kidney, one-clip (2K1C) is a model of renovascular hypertension where we previously found an exaggerated intracellular calcium (Ca²⁺_i) response to angiotensin II (ANG II) in isolated afferent arterioles (AAs) from the clipped kidney. To test if nitric oxide (NO) ameliorate the exaggerated ANG II response in 2K1C, we studied ANG II (10^-7 mol/L) induced calcium signaling and contractility with or without the NO synthase (NOS) inhibitor L-N(G)-Nitroarginine methyl ester (L-NAME). In AAs from the non-clipped kidney, L-NAME increased the ANG II induced Ca²⁺_i response from 0.28 ± 0.05 to 0.55 ± 0.09 (fura-II 340 nm/380 nm ratio), and increased contraction from 80 ± 6% of baseline to 60 ± 6% of baseline (p<0.05). In vessels from sham and clipped kidneys, L-NAME had no effect. In DAF-FM loaded AAs from the non-clipped kidney, ANG II increased NO-derived fluorescence to 145 ± 34% of baseline (p<0.05 vs. sham), but not in vessels from the sham or clipped kidney. Endothelial NOS (eNOS) mRNA and ser-1177 phosphorylation were unchanged in both kidneys from 2K1C, while eNOS protein was reduced in the clipped kidney compared to sham. Cationic aminoacid transferase-1 and 2 mRNAs were increased in 2K1C, indicating increased availability of L-arginine for NO synthesis, but counteracted by decreased scavenging of the eNOS inhibitor asymmetric dimethylarginine by dimethylarginine dimethylaminohydrolase2. In conclusion, the Ca²⁺_i and contractile response to ANG II is blunted by NO release in the non-clipped kidney. This may protect the non-clipped kidney from the hypertension and elevated ANG II levels in 2K1C.