The pathways by which pinazepam and diazepam are metabolized by rat microsomes in vitro were studied in detail. Using a gas chromatographic technique to measure either the two parent compounds or their metabolites, it was demonstrated that pinazepam and diazepam were metabolized through the same pathways, but at different rates. Pinazepam was mainly N-dealkylated, while the C3-hydroxylation was a minor metabolic pathway. Diazepam was mostly C3-hydroxylated, with very little being N-demethylated. The different rats for the two metabolic pathways could be of importance in differentiating the action of the two drugs in vivo.