Bretazenil is a partial agonist at diazepam-sensitive (DS) GABAAreceptors, and it also binds with high affinity to diazepam-insensitive (DI) GABAAreceptors. A unique discriminative stimulus effect transduced by binding at DI benzodiazepine (BZ) receptors has been reported in pigeons, but has not been established in rats. Further, differences have been observed between rats and pigeons in results of drug discrimination experiments utilizing BZ receptor partial agonists. Therefore, to examine the discriminative stimulus effects of bretazenil and to explore the possibility of species differences in substitution profiles, pigeons and rats were trained to discriminate 0.3 mg/kg bretazenil from vehicle. Fretazenil (0.03–1.0 mg/kg) did not substitute for bretazenil in pigeons, despite full substitution of bretazenil for flumazenil in this species. Flumazenil (0.03–10.0 mg/kg) also did not substitute for bretazenil in rats, despite the partial agonist effects of flumazenil in rats. Likewise, midazolam (0.3–1.0 mg/kg) did not substitute for bretazenil in pigeons, despite the fact that bretazenil partially substitutes for midazolam in pigeons. However in rats, midazolam produced full, dose-dependent substitution (0.03–3.2 mg/kg). Differences may result from different fractional receptor occupancy requirements for the mediation of discriminative stimulus effects throngh DS BZ receptors, and/or from a contribution of DI BZ receptor binding in pigeons.