In vitro lipophilicity of a series of benzodiazepines was evaluated by octanol: buffer partition ratio at physiological pH, and by retention time on a reverse-phase high-pressure liquid chromatographic (HPLC) system with a neutral-pH mobile phase. Both approaches ranked diazepam as highly lipophilic, but overall the two indices were poorly correlated (r = 0.23). For seven of the benzodiazepines, the in vivo volume of distribution (Vd) was determined in pharmacokinetic studies. After correlation for individual values of protein binding, Vd for unbound drug was significantly correlated with octanol: buffer partition ratio (r = 0.74), and to a greater extent with HPLC retention (r = 0.81). Thus, lipid solubility at least partly determines the extent of benzodiazepine distribution in vivo, which in turn is a major determinant of the duration of clinical action after single doses.