Administration of a new specific serotonin uptake inhibitor, fluoxetine, depressed the firing rate of raphe neurons. A highly significant increase in serum prolactin levels was observed after ip injection of 30 mg/kg of 5-hydroxytryptophan (5-HTP) in male or female rats pretreated with 10 mg/kg (ip) of fluoxetine. Neither 5-HTP nor fluoxetine given separately had any effect on serum prolactin levels. In animals pretreated with methysergide the combination of fluoxetine and 5-HTP did not increase significantly serum levels of prolactin. In addition, the serotonin agonist quipazine elevated significantly serum prolactin levels in male and female rats. The results of this study strengthen the idea that 5-HTP is acting via serotonin-containing neurons that influence anterior pituitary prolactin release, and that serotonin receptor activation leads to prolactin release.