Eight cyclosporine (CsA) metabolites were isolated from the urine of renal-transplant patients by high-pressure liquid chromatography. Structure and purity of the metabolites were assessed by fast atomic bombardment/mass spectroscopy, by proton nuclear magnetic resonance (NMR), and, when the quantity of metabolites permitted, by 13C-NMR. The immunosuppressive activities (I) of the metabolites were tested in three separate in vitro systems: primary and secondary mixed lymphocyte reactions as well as by a mitogen-stimulated system. The I, as measured by comparing the concentration of each metabolite required for 50% inhibition of incorporation of [3H] thymidine, varied among the assay systems, as did the ranking of I among the test systems. In general, the I of most metabolites in all assay systems were less than 10% of that for CsA. Metabolites with single modifications exhibited the greatest I; e.g., that of M-17 was congruent to 16% of that of CsA (potency ratio 0.16) in a secondary mixed lymphocyte reaction. The significance of these findings in relation to therapeutic monitoring of CsA is discussed.