Major depression is frequently associated with hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. Clinically effective therapy with antidepressant drugs normalizes the disturbed activity of HPA axis, in part, by decreasing corticotropin-releasing hormone (CRH) synthesis, but the mechanism of this action is poorly recognized. In order to find out whether antidepressants directly affect CRH gene promoter activity, we studied their effect on undifferentiated and differentiated Neuro-2A cells, and for comparison the effect of the selected antidepressants on AtT-20 cells was also determined. The cells were stably transfected with a human CRH promoter fragment (-663 to +124 bp) linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The regulation of CRH gene promoter activity is similar in Neuro-2A cells, both intact and differentiated, and in AtT-20 cell line, and cAMP/PKA-dependent pathway plays an important role in the stimulation of CRH gene. It was found that imipramine, amitryptyline, desipramine, fluoxetine, and mianserin, present in the culture medium for 5 days, in a concentration-dependent manner inhibited basal hCRH gene promoter activity in undifferentiated Neuro-2A cells, while other drugs under study (citalopram, tianeptine, moclobemide, venlafaxine, reboxetine, mirtazapine, and milnacipram) were inactive. In the differentiated cells, all examined antidepressants, except moclobemide (no effect) and tianeptine (increase), inhibited hCRH gene transcription. Moreover, in differentiated cells, the drugs acted stronger and were effective at lower concentrations. Forskolin-induced CAT activity was attenuated by imipramine and fluoxetine and to a lesser degree by amitriptyline and desipramine in differentiated cells, whereas other drugs were inactive. Moreover, imipramine and fluoxetine, but not tianeptine, showed moderate inhibitory effect on CRH gene promoter activity also in AtT-20 cell line, commonly used in CRH gene regulation studies. These results indicate that neuron-like differentiated Neuro-2A cells are a better model than pituitary and intact neuroblastoma to investigate the mechanism of psychotropic drug action. Inhibition of CRH gene promoter activity by antidepressant drugs may be a molecular mechanism by which these drugs inhibit the activity of HPA axis.