Melatonin, the major hormone produced by the pineal gland, is shown to have anticonvulsant effects. Nitric oxide (NO) is a known mediator in seizure susceptibility modulation. In the present study, the involvement of NO pathway in the anticonvulsant effect of melatonin in pentylenetetrazole (PTZ)-induced clonic seizures was investigated in mice. Acute intraperitoneal administration of melatonin (40 and 80 mg/kg) significantly increased the clonic seizure threshold induced by intravenous administration of PTZ. This effect was observed as soon as 1 min after injection and lasted for 30 min with a peak effect at 3 min after melatonin administration. Combination of per se non-effective doses of melatonin (10 and 20 mg/kg) and nitric oxide synthase (NOS) substrate L-arginine (30, 60 mg/kg) showed a significant anticonvulsant activity. This effect was reversed by NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg), implying an NO-dependent mechanism for melatonin effect. Pretreatment with L-NAME (30 mg/kg) and N(G)-nitro-L-arginine (L-NNA, 10 mg/kg) inhibited the anticonvulsant property of melatonin (40 and 80 mg/kg) and melatonin 40 mg/kg, respectively. Specific inducible NOS (iNOS) inhibitor aminoguanidine (100 and 300 mg/kg) did not affect the anticonvulsant effect of melatonin, excluding the role of iNOS in this phenomenon, while pretreatment of with 7-NI (50 mg/kg), a preferential neuronal NOS inhibitor, reversed this effect. The present data show an anticonvulsant effect for melatonin in i.v. PTZ seizure paradigm, which may be mediated via NO/L-arginine pathway by constitutively expressed NOS.