Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to PIP(2) where it recruits clathrin and stimulates lattice assembly. Murine PI5KIalpha has been shown to enhance endocytosis in a PIP(2) dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP(2) production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely due to a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP(2) binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP(2) levels and reduced apical surface expression of ENaC in CCD cells, down regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform specific siRNA resulted in a 4 fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP(2) production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.