Plasma binding of imipramine and lidocaine, two drugs that bind predominantly to alpha-1 acid glycoprotein (AAG), and of diazepam and phenytoin, albumin-bound drugs, were studied in fresh human plasma. Binding was determined by equilibrium dialysis with 0.1 M pH 7.4 phosphate buffer at 37 degrees C. AAG was determined by immunodiffusion. Lidocaine free fraction (FF) (30-35%) was time-dependent, reaching equilibrium by five hours. However, dialysis time exceeding 8 hours greatly increased lidocaine FF (60%) accompanied by a decrease in AAG concentration. Lidocaine binding and AAG-concentration were not affected by plasma freezing, and were independent of lidocaine concentrations from 0.5-10 micrograms/ml. Imipramine behaved comparably, with FF increasing from 15% at 5 hours of dialysis to 30% at 24 h, concurrent with a drop in AAG concentration. Imipramine binding and AAG concentration were also independent of plasma freezing and drug concentration from 0.25 to 10.0 micrograms/ml. Binding of diazepam (FF = 1.2%) and phenytoin (FF = 17%) were stable at 24 hours, not affected by sample freezing or drug concentration, and were independent of AAG concentration. Therefore, changes in AAG concentration alter plasma protein binding of AAG-bound drugs lidocaine and imipramine. Extended dialysis results in decreased concentrations of immunoreactive AAG and increased FF. In contrast, the binding of albumin-bound drugs diazepam and phenytoin are not affected by these variables.