An ultracentrifugation method was used for the determination of the tacrolimus (FK 506) free fraction in plasma. Samples of 400 microL were centrifuged for 6 h at 160,000 x g, and concentrations of [3H]tacrolimus were measured in the original plasma and in the second layer of the supernatant. The binding of tacrolimus in normal plasma averaged 73% and was independent of the drug concentration. Albumin (3.5%) binding averaged 44.4-57.0%, and alpha 1-acid glycoprotein (100 mg/L) binding averaged 39%. Hepatic transplant patients exhibited 72.0% +/- 3.6% binding of tacrolimus. Plasma protein binding is probably not an important factor in the disposition of tacrolimus.