Recent studies in vitro have shown that the cAMP response element-binding (CREB) co-activator, transducer of regulated CREB activity (TORC), is required for transcriptional activation of the corticotrophin-releasing hormone (CRH) gene. To determine the physiological importance of TORC2 regulating CRH transcription during stress, we examined the localisation of TORC2 in CRH neurones, as well as the relationship between changes in CRH heterogeneous nuclear (hn)RNA, nuclear translocation of TORC2 and binding of TORC2 to the CRH promoter. Immunohistochemistry revealed TORC2 immunoreactivity (irTORC2) in the dorsolateral (magnocellular) and dorsomedial (parvocellular) regions of the hypothalamic paraventricular nucleus (PVN). Although staining was mostly cytosolic under basal conditions, there was a marked increase in nuclear irTORC2 in the dorsomedial region after 30 min of restraint, concomitant with increases in CRH hnRNA levels. Levels of nuclear irTORC2 and CRH hnRNA had returned to basal 4 h after stress. Double-staining immunohistochemistry showed TORC2 co-staining in 100% of detected CRH neurones, and nuclear translocation after 30 min of restraint in 61%. Cellular distribution of TORC2 in the dorsolateral PVN was unaffected by restraint. Chromatin immunoprecipitation experiments showed recruitment of TORC2 and phosphorylated CREB (pCREB) by the CRH promoter after 30 min of restraint, but 4 h after stress only pCREB was associated with the CRH promoter. The demonstration that TORC2 translocates to the nucleus of hypothalamic CRH neurones and interacts with the CRH promoter in conjunction with the activation of CRH transcription during restraint stress, provides strong evidence for the involvement of TORC2 in the physiological regulation of CRH transcription.