The effects of chronic administration of the benzodiazepine receptor antagonist, flumazenil (Ro 15-1788; 4 mg/kg/day for 14 days in drinking water) on the performance of adult rats in the 12-arm radial maze were studied. Relative to controls, the animals treated with flumazenil showed an increase ( P < 0.002) in non-appetitively motived exploratory behavior, so called because it occurred in 88% of instances in non-baited alleys, facing the well-illuminated “enriched environment” of the center of the room, as opposed to the baited alleys, facing the “dull” comer of the room. This behavior emerged between day 5 and 7 of treatment with the drug, it continued to increase over the period of treatment with drug ( P < 0.002), and reached its peak at day 3, after withdrawal of the drug ( P < 0.008; a longer duration was not investigated). The occurrence of non-appetitively motivated exploratory behavior was inversely correlated with the scores for urination/defecation ( P < 0.003) and, therefore, most likely reflected the anxiolytic action of flumazenil. During treatment with drug or vehicle, the control and the drug groups made comparable numbers of “working memory” errors ( P = 0.17). However, upon withdrawal of drug and introduction of alley gates (to confine the animal for 10 sec to the center platform, after an alley was explored), the working memory errors of the rats exposed to the drug, remained unchanged ( P = 0.35), relative to the preceding three trials, while the performance of the control group was disrupted, as shown by an increase in the numbers of errors ( P < 0.004). At day seven of treatment with drug, the emergence of exploratory behavior was associated with an increased density and/or affinity of benzodiazepine receptors in cortex, hippocampus and brain stem, while three days after withdrawal of drug, when the exploratory behavior reached its peak, there was a reduction in GABA-enhanced binding of [ 3H]flunitrazepam in the cortex.