224 Cyclosporine (CsA) inhibits calcineurin (CN), a Ca2+/calmodulin-dependent phosphatase widely distributed in tissues. Since CsA distributes to many tissues, we investigated the extent of CN inhibition in different mouse tissues when CsA is added to tissues in vitro and when CsA is administered by oral dosing in vivo. For in vitro experiments, tissue homogenates were analyzed for CN activity with CsA concentrations in the assay of 0.1 to 10,000 ng/ml. For in vivo experiments, groups of 4 male BALB/c mice were gavage-fed CsA (Neoral) 100 mg/kg and tissues were studied at 0-72 hr. CsA serum levels were measured by EMIT assay. CN activity (%peptide hydrolyzed/min/mg protein) was highest in brain (200-300), intermediary in kidney, spleen, heart, and testis (10-20), and lowest in liver (<10). Comparison with CNAα knockout mice proved that >80% CN activity in each tissue was CNAα. To compare in vivo to in vitro, CsA concentration and IC50 for CN inhibition were expressed per gram of tissue and also per ml of tissue homogenate. (Table)TableTissues which concentrated CsA in vivo showed good agreement between in vivo and in vitro estimates of IC50 when corrected for assay dilution. Liver and kidney had the highest CsA concentrations and the greatest effect of CsA. Brain and testis had low CsA levels. Part of CN in brain (80%) and heart (40%) was absolutely resistant to saturating CsA because of inadequate cyclophilin: adding cyclophilin to the assay overcame resistance. Thus differential organ effects of CsA may reflect differences in tissue penetration by drug, abundance of cyclophilin, and abundance of CN. Sensitivity of kidney to CsA and resistance of heart and testis may be explained on this basis. In contrast, brain is resistant both to CsA accumulation and CN inhibition, leaving neurotoxicity unexplained. Research supported by Novartis, MRC, and KFC.