AbstractThe endocannabinoid system is an important regulator of the hormonal and behavioral stress responses, which critically involve corticotropin‐releasing factor (CRF) and its receptors. While it has been shown that CRF and the cannabinoid type 1 (CB1) receptor are co‐localized in several brain regions, the physiological relevance of this co‐expression remains unclear. Using double in situ hybridization, we confirmed co‐localization in the piriform cortex, the lateral hypothalamic area, the paraventricular nucleus, and the Barrington's nucleus, albeit at low levels. To study the behavioral and physiological implications of this co‐expression, we generated a conditional knockout mouse line that selectively lacks the expression of CB1 receptors in CRF neurons. We found no effects on fear and anxiety‐related behaviors under basal conditions nor after a traumatic experience. Additionally, plasma corticosterone levels were unaffected at baseline and after restraint stress. Only acoustic startle responses were significantly enhanced in male, but not female, knockout mice. Taken together, the consequences of depleting CB1 in CRF‐positive neurons caused a confined hyperarousal phenotype in a sex‐dependent manner. The current results suggest that the important interplay between the central endocannabinoid and CRF systems in regulating the organism's stress response is predominantly taking place at the level of CRF receptor‐expressing neurons.