TRIM, a selective neuronal NOS inhibitor, had anxiolytic effects in the elevated plus-maze (EPM) test. The aim of the present study was to evaluate the involvement of serotonergic system in the anxiolytic-like effect of TRIM in the EPM test, a widely used animal model of anxiety. The anxiolytic-like effect of TRIM (50 mg/kg, i.p.) in adult Wistar albino male rats in the EPM test was antagonized by pretreatment with the 5-HT depleting agent; parachlorophenylalanine methyl ester (3 × 150 mg/kg i.p.) that inhibits 5-HT synthesis; methiothepin (0.1 mg/kg, i.p.), a nonselective 5-HT receptor antagonist; WAY 100635 (0.1 mg/kg i.p.), a selective 5-HT1A receptor antagonist; GR 127935 (3 mg/kg i.p.), a selective 5-HT1B/1D receptor antagonist; cyproheptadine (3 mg/kg i.p.), a 5-HT2 receptor antagonist; or ketanserin (5 mg/kg i.p.), a 5-HT2A/2C receptor antagonist. The anxiolytic-like effects of TRIM thus appear to be mediated in part by 5-HT1 and 5-HT2 receptors.