Buspirone, a 5-HT (5-hydroxytryptamine, serotonin)(1A) partial agonist, is being used as an anxiolytic drug. The mechanism of action is explained by an effect on the 5-HT system. The main source of 5-HT in the forebrain is the dorsal raphe nucleus (DRN). However, there are also other populations of non-5-HT neurons in the DRN. Here, we investigated the effect of acute and chronic buspirone treatments on the 5-HT and non-5-HT cells, the neuronal nitric oxide synthase (nNOS) and tyrosine hydroxylase (TH) cells, in the DRN. Rats received either an acute or chronic administration of buspirone or saline. Hereafter, the brains were processed for 5-HT, nNOS, and TH immunohistochemistry. We found that acute and chronic buspirone treatments significantly lowered the mean optical density of nNOS in the DRN as compared to controls. Meanwhile only the chronic buspirone treatment reduced the mean density of 5-HT and TH immunoreactivity but not the acute buspirone as compared to saline treated animals. Our findings suggest that buspirone treatment affects not only the intracellular content of 5-HT but also nNOS and TH. Therefore, the cellular effect of buspirone is more complex than thought.