Eight cyclosporine (CsA) metabolites were isolated from the urine of renal transplant recipients. The structure and purity of the metabolites were characterized by fast atom bombardment/mass spectroscopy as well as by proton and 13C nuclear magnetic resonance. The in vitro toxicity of the metabolites were tested using a porcine renal epithelial cell line (LLC-PK1). None of the metabolites was as effective as CsA in inhibiting cell growth and DNA, RNA, or protein synthesis, with the majority of them exhibiting activity less than 10% of that of CsA when the IC50 (the concentration required for 50% inhibition of that particular metabolic function) values were compared. The exception to this was the demethylated metabolite M-21, which exhibited a potency of 17-50% of CsA for the various metabolic parameters examined. The results suggest that the immunosuppressive activity of metabolites may be dissociated from their toxicity. Morphologically, CsA and the metabolite M-21 resulted in changes consistent with the vacuolization seen in tubular cells exposed to CsA in vivo. In contrast, M-17 up to the maximum concentration tested (25,000 micrograms/L) was found not to cause such changes.