Thromboxane (TxA2) and nitric oxide (NO) are potent vasoactive autocoids that modulate tubuloglomerular feedback. Each is produced in the macula densa (MD) by cyclooxygenase-2 (COX-2) and neuronal nitric oxide synthase (nNOS) respectively. Both enzymes are similarly regulated and their interaction may be an important factor in the regulation of TGF and glomerular filtration rate (GFR). We tested the hypothesis that TGF is modified by the balance between MD nNOS-dependent NO and MD COX-2-dependent TxA2. We measured maximal TGF during perfusion of the loop of Henle (LH) by continuous recording of the proximal tubule stopped flow pressure (PSF) response to LH perfusion of artificial tubular fluid (ATF) at 0 and 40 nl/min. The response to inhibitors of COX-1 (SC-560), COX-2 (paracoxib, Pxb) and nNOS (L-NPA) added to the ATF solution was measured in separate nephrons. COX-2 inhibition with Pxb reduced TGF by 46% (ATF + Veh: vs ATF + Pxb), whereas COX-1 inhibition with SC-560 reduced TGF by only 23%. Pretreatment with intravenous infusion of SQ-29,548, a selective TPR antagonist blocked all of the SC-560 effect on TGF, suggesting that this effect was due to activation of TPR. However SQ-29,548 only partially diminished the effect of Pxb (-66%). Specific inhibition of nNOS with L-NPA increased TGF, as expected. However the ability of Pxb to reduce TGF was significantly impaired with co-microperfusion of L-NPA. These data suggest that COX-2 modulates TGF by two pro-constrictive actions: generation of TxA2 acting on TPR and by simultaneous reduction of NO.