Mouse epidermal cytosol contains a protein phosphatase with Mr 38,000, which dephosphorylates the elongation factor 2 (EF-2) of protein biosynthesis and is stimulated after topical application of TPA to mouse skin [(1988) Biochem. Biophys. Res. Commun. 153, 1129-1135]. Dephosphorylation of EF-2 by this phosphatase is inhibited by okadaic acid at concentrations as low as 10(-8) M, but not by heparin up to concentrations of 600.micrograms/ml. The catalytic subunit of protein phosphatase 2A (PP2Ac) with EF-2 as a substrate exhibits the same sensitivity towards okadaic acid and insensitivity towards heparin as the EF-2 phosphatase of epidermal cytosol. The catalytic subunit of protein phosphatase 1 (PP1c) is strongly suppressed by heparin and less sensitive towards okadaic acid than PP2Ac. PP2Ac is around 50 times more efficient in dephosphorylating EF-2 than PP1c. These data indicate that the TPA-stimulated EF-2 phosphatase in epidermal cytosol is a type 2A protein phosphatase.