Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase expressed at high levels in brain. The immunosuppressive drugs cyclosporin A and FK506, but not rapamycin are specific inhibitors of calcineurin, the inhibitory effects of which have been elucidated in the immune system. Here by using these compounds as inhibitors, we assayed the enzyme in mouse brain after injection of 12.5 nmol cyclosporin A, FK506, or rapamycin into the left lateral ventricle of mouse brain. Data from calcineurin activity assay suggest that infusion of cyclosporin A or FK506, rather than rapamycin inhibited calcineurin activity in brain and in a substrate noncompetitive manner, which is revealed by the in vitro enzyme kinetic analysis. Cyclosporin A or FK506 injected into brains also affected the inhibitory effects of cyclosporin A or FK506 added to brain extracts on calcineurin activity. The results may be ascribed to the decreased free immunophilin in brain after infusion of corresponding immunosuppressant, or the fact that two immunophilin-immunosuppressant complexes have not completely identical interaction sites on calcineurin.