Parathyroid hormone (PTH) inhibits Na+-K+-ATPase activity by serine phosphorylation of the α1-subunit through ERK-dependent phosphorylation and translocation of protein kinase Cα (PKCα). On the basis of 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 cells, a model of renal proximal tubule, PTH-stimulated ERK phosphorylation and membrane translocation of PKCα were prevented by inhibition of Src kinase, PLC, and calcium entry. Pharmacological inhibition of PLA2 did not prevent PTH-stimulated ERK phosphorylation but completely prevented PKCα translocation. Silencing the expression of cytosolic or calcium-independent PLA2 also prevented PTH-mediated phosphorylation of Na+-K+-ATPase α1-subunit and PKCα without blocking ERK phosphorylation. Inhibition of Na+-K+-ATPase activity by the PLA2 metabolites arachidonic acid and 20-hydroxyeicosatetraenoic acid was prevented by specific inhibition of PKCα but not by U0126, a MEK-1 inhibitor. Transient transfection of constitutively active MEK-1 cDNA induced phosphorylation of Na+-K+-ATPase α1-subunit and PKCα, 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 PKCα.