Disopyramide is an antiarrhythmic drug that exhibits nonlinear binding to plasma proteins. As a result, the total body clearance increases with increasing total drug plasma concentration. A rapid and sensitive method for the determination of free (unbound) disopyramide plasma concentrations is described. The procedure employs an ultrafiltration system (Centrifree), which can be used for basic drugs, along with an enzyme multiplied immunoassay system (EMIT) for the measurement of free disopyramide concentrations in plasma water filtrate. The EMIT method was adapted to permit measurement of disopyramide in plasma over a concentration range of 0.02-1.2 micrograms/ml. Plasma storage at -20 degrees C, filtration volume, or the presence of buffer and mono-N-dealkylated metabolite in plasma did not affect the binding determinations. There was no loss of drug during the filtration process. A good correspondence was found between the EMIT assay and a high performance liquid chromatography method, when applied to plasma samples obtained from a human subject who had ingested disopyramide. Furthermore, the extent of protein binding determined by the ultrafiltration system and by equilibrium dialysis were in good agreement. The binding of disopyramide in fortified human plasma decreased from 64 to 52% over a total drug concentration range of 1-5 micrograms/ml.