In the present study we used LLC-PK1 cells, a porcine renal proximal tubular cell line, to investigate whether PI3 kinase activation was involved in the anti-apoptotic effect of ouabain, a specific inhibitor of Na,K-ATPase, Apoptosis was induced by actinomycin D (Act D, 5 muM) and assessed by appearance of hypodiploid nuclei and DNA fragmentation. Ouabain attenuated Act D-induced apoptotic response in a dose-dependent manner. Incubation in a low K+ medium (0.1 mM) which is another way to decrease Na,K-ATPase activity also had anti-apoptotic effect. Both ouabain and low K+ medium increased the PI3 kinase activity in p85 immunoprecipitates, Ouabain, as well as incubation in the low K+ medium, also increased the phosphorylation of Akt, Inhibition of PI3 kinase by either wortmannin or LY294002 reversed the cytoprotective effect of ouabain, These data together indicate that inhibition of Na,K-ATPase activates PI3 kinase in LLC-PK1 cells which could then exert the cytoprotective effect.