The involvement of nitrergic mechanisms in the behavioural effects of withaferin A 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 withaferin A (40 mg/kg, i.p.) in the elevated plus-maze, whereas the inactive enantiomer D-arginine (100 mg/kg) did not. Neither withaferin A alone nor in combination with L- or Darginine 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 withaferin A (40 mg/kg) in the rotarod test. Withaferin A (40 mg/kg i.p.) did inhibit the brain NO synthase activity measured ex vivo by NOx assay. We conclude that a suppression of NO synthase activity may be important in the anxiolytic-like effect of withaferin A. © 2011 Academic Journals.