The binding of diazepam (DZ) and its major metabolite desmethyldiazepam (DMDZ) to plasma protein was evaluated in a series of controlled in vitro studies using equilibrium dialysis. Free fraction (FF) of both drugs alone changed significantly with total plasma drug concentration, but the increased FF (reduction in binding) did not occur until concentrations considerably exceeded those encountered during typical therapeutic use. Increasing concentrations of one drug at a time tended to increase FF for the other, although the effects were, at most, of borderline significance. Simultaneously increasing concentrations of both drugs led to significantly increased FF for both. Thus, DZ and DMDZ appear to bind to the same site or sites on plasma albumin. Binding is concentration-independent within and considerably above the usual therapeutic range.