Eight to 14 plasma samples were obtained from 12 patients on chronic prednisone treatment following their usual dose of prednisone (0.262 to 1.053 mg/kg . day) and following an equivalent amount of prednisolone given i.v. Total and unbound prednisolone were assessed by high performance liquid chromatography and by equilibrium dialysis. The immunosuppressive activity of the plasma was determined as the inhibition of the mixed lymphocyte reaction. The area under the plasma concentration time curve (AUC) for total and unbound prednisolone and the area under the inhibition time curve (AUIC) of the mixed lymphocyte reaction were determined by the trapezoidal rule. A hyperbolic concentration response curve was found when inhibition of the mixed lymphocyte reaction was plotted versus concentrations of total and free prednisolone. The mean EC50 (+/- SD) for total prednisolone concentration following the oral and the i.v. dosing was 66.3 +/- 26.7 and 86.5 +/- 30.9 ng/ml, respectively; for free prednisolone concentration the corresponding values were 10.0 +/- 5.0 and 12.4 +/- 12.6 ng/ml. A similar hyperbolic relationship is observed when the AUIC is plotted versus the AUC of free or total prednisolone, but not when AUIC is plotted versus the oral dose. Over the range of drug concentrations observed, no difference is found between total and free concentrations in the ability to predict inhibition of the mixed lymphocyte reaction.