Parathyroid hormone (PTH) inhibits Na⁺, K⁺ ATPase activity by serine phosphorylation of the ₁ subunit through ERK-dependent phosphorylation and translocation of protein kinase C- (PKC). Based on previous studies we postulated that PTH regulates sodium pump activity through Src kinase, PLC, and calcium-dependent ERK phosphorylation. In the present work utilizing opossum kidney (OK) cells, a model of renal proximal tubule, PTH-stimulated ERK phosphorylation and membrane translocation of PKC was prevented by inhibition of Src kinase, PLC, and calcium entry. Pharmacologic inhibition of PLA₂ did not prevent PTH-stimulated ERK phosphorylation but completely prevented PKC translocation. Silencing the expression of cPLA₂ or iPLA₂ also prevented PTH-mediated phosphorylation of Na⁺, K⁺ ATPase 1-subunit and PKC without blocking ERK phosphorylation. Inhibition of Na-K ATPase activity by the PLA₂ metabolites, arachidonic acid and 20-HETE, was prevented by specific inhibition of PKC but not by U0126, a MEK-1 inhibitor. Transient transfection of constitutively active MEK-1 cDNA induced phosphorylation of Na⁺, K⁺ ATPase ₁-subunit and PKC which was prevented by PLA₂ inhibition. We conclude that PTH stimulates Na⁺, K⁺ ATPase phosphorylation and decreases the activity of Na⁺, K⁺ ATPase by a sequential activation of a signaling pathway involving Src kinase, PLC, ERK, PLA₂ and PKC.