Previous studies have shown that L-type Ca²⁺channels (LCC) blockers primarily dilate resting and angiotensin II (Ang II) constricted afferent arterioles (AA), but do not influence either resting or Ang II constricted efferent arterioles (EA). In contrast, blockade of T-type Ca²⁺channels (TCC) dilate EA and prevent Ang II mediated efferent constriction. The present study determined the role of LCC and TCC in mediating the AA and EA constriction following inhibition of nitric oxide synthase (NOS) and tested the hypothesis that inhibition of NOS increases the influence of LCC on EA. Using the isolated blood-perfused rat juxtamedullary nephron preparation, single AA or EA were visualized and superfused with a NOS inhibitor, N -nitro-L-arginine (L-NNA), with or without concomitant treatment with an LCC blocker, diltiazem or a TCC blocker, pimozide. In response to L-NNA (1, 10 and 100µmol/L), AA and EA diameters decreased significantly by 6.0±0.3, 13.7±1.7 and 19.9±1.4%, and by 6.2±0.5, 13.3±1.1 and 19.0±1.9%, respectively. During TCC blockade with pimozide (10µmol/L), LNNA did not significantly constrict afferent (0.9±0.6, 1.5±0.5 and 1.7±0.5%) or efferent (0.4±0.1, 2.1±0.7 and 2.5±1.0%) arterioles. In contrast to the responses with other vasoconstictors, the L-NNA- induced constriction of EA, as well as AA, was reversed by diltiazem (10µmol/L). The effects were overlapping since pimozide superimposed on diltiazem did not elicit further dilation. When the effects of L-NNA were reversed by superfusion with an NO donor, SNAP (10µmol/L), diltiazem did not cause significant efferent dilation. As a further test of LCC activity, a 55 mmol/L KCL, which depolarizes and constricts AA, caused only a modest constriction in resting EA (8.7±1.3%), but a stronger EA constriction during concurrent treatment with L-NNA (23.8±4.8%). In contrast, norepinephrine caused similar constrictions in both L-NNA treated and non-treated arterioles. These results provide evidence that NO inhibits LCC and TCC activity and that NOS inhibition mediated arteriolar constriction involves activation of LCC and TCC in both AA and EA. The difference in responses to high KCL between resting and L-NNA constricted EA, and the ability of diltiazem to block EA constriction caused by L-NNA contrasts with the lack of efferent effects in resting and SNAP-treated L-NNA preconstricted arterioles and during Ang II mediated vasoconstriction suggesting a recruitment of LCC in EA when NOS is inhibited. These data help explain how endothelial dysfunction associated with hypertension may lead to enhanced activity of LCC in post-glomerular arterioles and increased post-glomerular resistance.