We used Western blot to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of 110 kDa subunit (p110) of PI3K in comparison to rats on a normal K diet. Since low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H₂O₂ stimulates the expression of p110 in M1 cells. Inhibition of PI3K with either wortmannin or LY294002 significantly increased channel activity in the CCD from rats on a K deficient (KD) diet or on a normal K diet. The stimulatory effect of wortmannin on ROMK channel activity can not be mimicked by inhibition of phospholipase C with U73122. This suggests that the effect of inhibiting PI3K was not the result of increasing phosphatidylinositol (PI) -4,5 phosphate ( PIP2) level. Moreover, application of exogenous PI-3,4,5 phosphate (PIP3) analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased the SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase the ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.