To investigate the phenomenon of different erythrocyte saturation capacities for cyclosporine (CsA) in the blood of different individuals, hemolysates of washed red cells were examined for the presence of a CsA-binding protein. Using gel filtration column chromatography of hemolysates from patients receiving CsA orally, the majority of erythrocyte-associated CsA eluted as a single peak with Mr 15,000-17,000, distinct from hemoglobin and carbonic anhydrase. [3H]CsA added to a hemolysate in vitro eluted similarly. [125I]CsA added to a hemolysate eluted much later in the same position as [3H]CsA mixed with albumin and myoglobin (presumably as free unbound drug). These findings indicate that CsA normally binds to an intraerythrocytic protein similar in molecular size to calf thymus cyclophilin (Mr 15,000). By equilibrium dialysis, the purified erythrocyte proteins calmodulin (Mr 16,700) and cytochrome b5 (Mr 15,000) failed to bind CsA. By equilibrium dialysis, [3H] CsA did bind to column fractions containing the CsA-binding protein, but [125I]CsA did not, suggesting that attachment to CsA occurs at or near a carbon-carbon double bond in an unusual nine-carbon amino acid of CsA. These results have important implications for CsA therapy with regard to distribution space, pharmacokinetics, and a possible protein-receptor mechanism of action.