Cyclosporine A (CsA) is extensively metabolized in the liver. Some of the known metabolites share immunosuppressive properties with the parent drug. Furthermore, CsA therapy is used in an increasing number of clinical conditions, some of which affect the pharmacokinetics of the drug. At this time, it is not yet known if CsA or its metabolites are responsible for the nephrotoxicity or hepatotoxicity observed in some individuals. Some high-performance liquid chromatography (HPLC) and monoclonal immunoassay procedures measure parent drug and not the pharmacologically active metabolites, while polyclonal immunoassays and nonspecific monoclonal antibody immunoassays measure both parent drug and metabolites. However, it is unlikely that the degree of cross-reactivity with metabolites correlates with their immunosuppressive effect. To overcome these drawbacks, we have developed a method of measuring CsA activity in whole blood using specific receptors from the cytosol of human mononuclear leukocytes that have been semipurified through ultracentrifugation. The basis of the procedure is the competitive binding at specific receptor(s) between a constant concentration of [3H]dihydrocyclosporine and the variable concentrations of cyclosporine and its pharmacologically active metabolites in whole blood. Comparisons between six different assays (three specific, two nonspecific, and the receptor assay) were made. Overall, the two specific radioimmunoassay procedures correlated well to HPLC, while correlation of the two nonspecific immunoassay procedures to HPLC was poor. Poor correlation was also found to exist between the radioreceptor assay and the nonspecific assays, indicating that the cytosolic binding of CsA and its metabolites cannot be correlated to currently available assays.