Ca 2 + -mobilizing and cAMP-dependent hormones rapidly increase sodium, potassium-dependent adenosine triphosphatase (Na + /K + -ATPase)-mediated transport in rat hepatocytes. To explore the possible role of protein phosphatases in these responses we used a protein phosphatase inhibitor, okadaic acid. Okadaic acid stimulation of ouabain-sensitive 8 6 Rb + -uptake was maximal between two and three minutes and displayed an EC 5 0 of 41 +/- 1 nM. Inhibition of Na + /H + exchange with an amiloride analog abolished the response to insulin, but had no effect on okadaic acid-mediated stimulation of Na + /K + -ATPase transport. In hepatocytes metabolically-radiolabeled with 3 2 P i , okadaic acid stimulated the incorporation of radioactivity into several 95 kDa peptides, one of which reacted with anti-LEAVE peptide antisera, that recognizes Na + /K + -ATPase α-subunits. In other experiments Na + /K + -ATPase was immunoprecipitated from detergent-solubilized membrane fractions of metabolically-radiolabeled cells with an antisera to purified rat kidney Na + /K + -ATPase. A 95 kDa phosphoprotein was immunoprecipitated using anti-Na + /K + -ATPase antisera, but not by preimmune serum. Okadaic acid stimulated incorporation of radioactivity into this band by 220 +/- 28%. These findings provide support for the hypothesis that rapid stimulation of hepatic Na + /K + -ATPase by hormones may be related to protein kinase/phosphatase-mediated changes in the phosphorylation state of the Na + /K + -ATPase α-subunit.