The Ca(2+)-dependent protein phosphatase activity of crude rat brain extracts measured in the presence of okadaic acid, exhibits the characteristic properties of the calmodulin-stimulated protein phosphatase, calcineurin. It is stimulated more than 200-fold by Ca2+ and inhibited by the calmodulin-binding peptide, M13, and by the immunosuppressive drug, FK506. It is insensitive to rapamycin at concentrations up to 1 microM. Its specific activity, based on calcineurin concentration determined by quantitative analysis of Western blots exposed to anti-bovine brain IgG, is ten to twenty times that of purified rat brain calcineurin assayed under similar conditions. Unlike the purified enzyme it is rapidly and irreversibly inactivated in a time-, temperature-, and Ca2+/calmodulin-dependent fashion without evidence of extensive proteolytic degradation. The enzyme is converted to a state which does not lose activity by removal of low molecular weight material by gel filtration. Reconstitution of a labile enzyme is achieved by the addition of the low molecular weight-containing fraction eluted from the gel filtration column. These observations indicate that calcineurin in crude brain extracts is under the control of Ca2+/calmodulin-dependent positive and negative regulatory mechanisms which involve unidentified endogenous factor(s).