A reproducible ultracentrifugation method has been developed for the measurement of the fraction of cyclosporin (CyA) unbound in plasma. The sample is centrifuged to remove any particulate matter, ultracentrifuged in polyallomer tubes and then frozen in liquid nitrogen. Appropriate sections are then cut from the tube for determination of the concentration of radioactivity and calculation of fraction drug unbound. Using this method, the fraction unbound has been measured in plasma from renal transplant patients receiving CyA and found to range between 0.04 to 0.122. The binding is temperature-dependent and principally hydrophobic.