The involvement of nitrergic mechanisms in the behavioural effects of diazepam in rats was studied in the elevated plus-maze, open-field and rotarod tests. Administration of the nitric oxide (NO) precursor L-arginine (100 mg/kg, i.p.), assumed to increase the synthesis of NO, abolished the anxiolytic-like effect of diazepam (2 mg/kg, i.p.) in the elevated plus-maze, whereas the inactive enantiomer D-arginine (100 mg/kg) did not. Neither diazepam alone nor in combination with L- or D-arginine affected the exploratory activity of animals in the open field. Pretreatment with L-arginine (100 and 200 mg/kg) did not modify the motor impairment of rats after diazepam (3 mg/kg) in the rotarod test. Diazepam (2 mg/kg i.p.) did not inhibit the cortical or hippocampal cytosolic NO synthase activity measured ex vivo by [3H]L-arginine assay. Diazepam was similarly ineffective in in vitro studies at concentrations up to 10 microM. We conclude that a suppression of NO synthase activity may be important in the anxiolytic-like effect of benzodiazepines. However, diazepam does not inhibit NO synthase directly, but may affect NO synthase activity indirectly via some unknown mechanism.