Effect of chronic administration of benzodiazepine (BZ) receptor antagonist Ro 15-1788 (flumazenil) (4 mg/kg once daily for 14 days) treatment and its withdrawal on locomotor activity, body temperature, and the binding pattern of receptor ligands that bind to GABA-BZ receptor ionophore complex in different regions of the brain of the rat was studied. Ro 15-1788 (× 14 d) increased the specific binding of [3H]ethyl-8-fluoro-5-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5α][1,4]benzodiazepine-3-car☐ylate ([3H]Ro 15-1788), [3H]ethyl-8-azido-5-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5α][1,4]benzodiazepine-3-car☐ylate ([3H]Ro 15-4513), [3H]flunitrazepam, and[35S]t-butylbicyclophosphorothionate ([35S]TBPS) in cerebral cortex, and this increase in binding remained upregulated during the drug withdrawal at 24. The binding of [3H]Ro 15-1788 was also found significantly increased in the hippocampus, but not in cerebellum and striatum. The chronic Ro 15-1788 treatment did not alter the specific binding of [3H]GABA. Rosenthal analysis of the saturation isotherms indicated that the observed upregulation in the binding pattern of [3H]Ro 15-1788 and [3H]Ro 15-4513 in the cerebral cortex was due to an increase in the binding capacity (B max ). The receptor affinity (K d ) was not changed. The withdrawal of Ro 15-1788 following its chronic administration also enhanced locomotor activity. However, no apparent change in body temperature was observed either due to chronic treatment or withdrawal. These data indicate that chronic Ro 15-1788 treatment and its withdrawal may produce an upregulation of subunits which bind the positive (benzodiazepines), negative (inverse agonist), and neutral (antagonist) ligands of benzodiazepine receptor. This may have an implication in drug discontinuation syndrome due to benzodiazepines.