To delineate the microvascular role of cyclooxygenase-2 (Cox-2) in modulating tubuloglomerular feedback (TGF) signals and to determine its relationship to neuronal nitric oxide synthase (nNOS), afferent (AA) and efferent (EA) arteriolar diameters of rat kidneys were assessed using the blood-perfused juxtamedullary nephron technique. The Cox-2 inhibitor NS-398 (10 μM) did not alter AA diameters in untreated kidneys but significantly constricted AAs by 17.0 ± 2.2% in kidneys treated with 10 mM acetazolamide, which enhances TGF-mediated AA constriction by increasing distal volume delivery. The NS-398-induced AA constriction was prevented after interruption of distal delivery by transection of the loops of Henle. The effect was selective for AAs since NS-398 did not influence EAs of untreated or acetazolamide-treated kidneys. Pretreatment with the nNOS inhibitor S- methyl-L-thiocitrulline (10 μM) prevented the NS-398-induced AA constriction observed during acetazolamide treatment. Although we previously demonstrated that acetazolamide treatment enhanced AA constrictor response to S-methyl-L- thiocitrulline, the enhancement by acetazolamide was inhibited by pretreatment with 10 μM NS-398 (16.4 ± 1.9 and 15.0 ± 0.5% with and without acetazolamide, respectively, P > 0.05). These results indicate that, during increased activation of TGF-dependent vasoconstrictor signals, Cox-2 generates vasodilatory metabolites in response to increased nNOS activity and thus participates in the counteracting modulation of TGF-mediated AA constriction.