Abstract: The raphe-hippocampal serotonin (5-HT) system is involved in the regulation of the hypothalamuspituitary-adrenal axis. The purpose of this study was to determine and compare the roles of 5-HT in the regulation of glucocorticoid receptor (GR) binding in the raphe nuclei and in the hippocampus. The effects of 5-HT, 5-HT agonists, and the 5-HT reuptake inhibitor citalopram on GR binding sites were studied in primary cultures of the fetal raphe nuclei and the hippocampus. Exposure of hippocampal cells to 5-HT, (±)-2,5-dimethoxy-4-iodoamphetamine (DOI; a 5-HT2 agonist), or citalopram resulted in an increase in number of GR binding sites. The effect of DOI was blocked by ketanserin (a 5-HT2 antagonist). Specific and saturable GR binding was found in raphe cells. Exposure of raphe cells to 5-HT, (±)-8 hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; a 5-HT1A agonist), or citalopram induced a significant decrease in number of GR binding sites. The effect of 8-OH-DPAT was reversed by WAY 100135 [N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionamide; a 5-HT1A antagonist]. These results show that the regulation of GRs during fetal life is structure-dependent and involves different 5-HT receptor subtypes. Moreover, the regulation of hippocampal GRs by citalopram suggests an action of antidepressants independent of their effects on monoamines.