Protein binding of tacrolimus (FK506) in human plasma was re-evaluated by equilibration dialysis and compared with that of FK506 previously reported by two different methods (about 99 and 77% by an ultrafiltration and ultracentrifugation method, respectively). The binding determined in this study was about 99% irrespective of FK506 concentrations added (0.5-10 ng/ml) and this value was very close to that estimated by the ultrafiltration method. The effect of mycophenolic acid (MPA, an active form of the immunosuppressant mycophenolate mofetil) and its glucuronide (MPAG, a major metabolite of mycophenolate mofetil in human plasma) on the binding was studied at concentration levels of 1 and 10 ng/ml of FK506. The binding was not affected significantly by the addition of MPA (25-100 micrograms/ml) and/or MPAG (100-1500 micrograms/ml). FK506 has already been reported not to cause significant changes of plasma protein binding of MPA. The results indicate that the unbound fraction of FK506 is about 1% in human plasma and that concomitant administration of FK506 and mycophenolic mofetil does not cause the drastic change of the binding of FK506 and MPA with human plasma proteins.