The biological effects of the Corticotropin-releasing factor (CRF) family of neuropeptides are mediated by mobilization of [Ca2+]. Aim of the current work was to examine if the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway is involved in the effect of CRF peptides in catecholamine synthesis and secretion from PC12 rat pheochromocytona cells, a model for the study of adrenal catecholamine production. PC12 cells express both types of CRF receptors. Our data are as follows: (a) The calcineurin inhibitor cyclosporine A (CsA) blocked norepinephrine secretion induced by ligands of either CRF type 1 (CRF1) or 2 (CRF2) receptors on PC12 cells. (b) Silencing NFAT2 expression using a selective NFAT2 siRNA blocked CRF1 and CRF2-induced NE production. (c) CRF ligands induced NFAT transcriptional activity in cells transfected with a luciferase reporter construct controlled by NFAT binding elements (NFAT-Luc). (d) CsA completely blocked the stimulatory effect of CRF1 and CRF2 ligands on NFAT activity in NFAT-Luc transfected cells. (e) PKA, PKC, p38-MAPK, Tpl2, Ha-Ras, and AKT1 were crucial intermediates for both CRF1 and CRF2-induced NFAT activation. Interestingly, MEK1/2 and ERK1/2 were crucial only for the CRF2-induced NFAT activation. (f) p38-MAPK and Tpl2 were crucial intermediates for both CRF1 and CRF2-induced norepinephrine production, while AKT1 affected only CRF2-induced norepinephrine production. In conclusion, our data suggest that CRF1 and CRF2 ligands activate the transcription factor NFAT and its activation is prerequisite for CRF-induced catecholamine production from chromaffin cells. J. Cell. Physiol. 227: 1861–1872, 2012. © 2011 Wiley Periodicals, Inc.