It was previously reported that the nitric oxide synthase (NOS) inhibitor agmatine had anxiolytic and antidepressant-like effects in rodents (Aglawe et al Brain Res Bull 167:37, 2021). Since serotonergic (5-HT) neurons of the dorsal raphe nucleus (DRN), expressing NOS, had lower excitability than the non-NOS expressing ones (Gartside et al Eur J Neurosci 51:1881, 2020), the anxiolytic and antidepressant-like effects of agmatine might involve stimulation of 5-HT neurons of the DRN. The present study aimed to examine this hypothesis. Acute and chronic effects of agmatine were tested. In acute experiments, male Wistar rats were anesthetized with chloralhydrate, and glass electrodes were inserted into their DRN. After spontaneously active 5-HT neurons were identified, and their basal activity recorded for at least one minute, agmatine was intravenously administered in cumulative doses of 1- 3 mg/kg. In chronic experiments, rats were administered agmatine (40 mg/kg/day, intraperitoneally, for 14 days); control rats received saline. On day 15th, one hour after the last agmatine or saline injection, rats were anesthetized and the firing activity of 5-HT neurons in their DRNs was recorded. We found that acute agmatine stimulated 5-HT neurons in a statistically significant way. Correspondingly, the average firing rate of 5-HT neurons in the DRN of rats chronically treated with agmatine was higher than in the vehicle-treated controls. In summary, anxiolytic and antidepressant-like effects of agmatine are likely to be mediated via a mechanism involving stimulation of 5-HT neurons.This work was supported by the grants APVV-20-0202, VEGA-2/0057/22 and VEGA-2/0045/24.