Previous studies propose 20-hydroxyeicosatetraenoic acid (20-HETE), a major arachidonic acid metabolite of Cytochrome P-450 (CYP), as a possible mediator of Na⁺,K⁺-ATPase inhibition by Dopamine (DA). The aim of this study was to investigate the intracellular mechanisms involved in this effect and to elucidate the DA receptor associated with the 20-HETE pathway in the rat kidney. DA (10^-5 M) inhibited Na⁺,K⁺-ATPase activity in microdissected tubular segments to 59.4±3.8% of control activity. This response was suppressed by the CYP4A inhibitor 17-octadecynoic acid (10^-6 M), which had no effect per se, thus confirming the participation of CYP arachidonic acid metabolites in DA-induced Na⁺,K⁺-ATPase inhibition. We next examined whether 20-HETE is involved in the signaling pathways triggered by either D₁ or D₂ receptors. Neither fenoldopam nor quinpirole (D₁ and D₂ agonists respectively, both 10^-5M) modified Na⁺,K⁺-ATPase activity when tried alone. However, coincubation of a threshold concentration of 20-HETE (10^-9M) with fenoldopam resulted in a synergistic inhibition of Na⁺,K⁺-ATPase activity (66±2% of control activity), while 20-HETE plus quinpirole had no effect. Furthermore, 20-HETE (10^-9M) synergized with forskolin (10^-5M) and with the diacylglycerol analog 1-oleoyl-2-acetoyl-sn-glycerol (OAG, 10^-11M) (62.0±5.3 and 69.9±2.0% of control activity respectively), indicating a cooperative role of 20-HETE with the D₁-triggered pathways. In line, no additive effect was observed when OAG and 20-HETE were combined at concentrations which per se produced maximal inhibition (10^-6 M). These results demonstrate that the inhibition of Na⁺,K⁺-ATPase activity by DA in the proximal tubule may be the result of the synergism between 20-HETE and the D₁ signaling pathway.