Evidence implicates the endocannabinoid (eCB) system as a negative modulator of neural and endocrine responses to acute stressors. Recently, eCB signaling was also reported to contribute to habituation of hypothalamo-pituitary-adrenal (HPA) axis responses to repeated homotypic stress. The present studies were initiated to distinguish a potential role of eCB signaling in the expression vs. the acquisition of habituation of the HPA axis response to repeated stress. In each of three experiments, adult male Sprague Dawley rats were exposed to daily, 30-minute sessions of loud white noise (95 dB), which resulted in a progressive decrease in HPA axis response over successive days. Cannabinoid receptor 1 (CB1) antagonist AM251 (0.5, 1.0 or 2.0 mg/kg, i.p.) was used to examine the role of eCB signaling in homotypic stressor habituation and heterotypic (novel) stressor cross-sensitization of neuroendocrine activity. Pretreatment with high dose (2.0 mg/kg) AM251 before each of 7 consecutive, daily loud noise exposures (acquisition of habituation) resulted in potentiation of stress-induced HPA axis activation and disruption of habituation. After an 8th loud noise exposure without AM251 pretreatment, the same group of rats displayed a habituated plasma corticosterone (CORT) level similar to that of controls, indicating that CB1 receptor antagonist pretreatments did not disrupt the acquisition of habituation. In two additional experiments, rats acquired habituation to loud noise drug free, then lower doses of AM251 (0.5 and 1.0 mg.kg) were administered before a final exposure (expression of habituation) to the homotypic stressor and/or a novel heterotypic stressor. CB1 receptor antagonism disrupted the expression of CORT response habituation and some of the c-fos mRNA reduction associated with it and facilitated novel stressor sensitization in doses that did not potentiate acute responses to these stressors. Collectively, these data suggest a progressive intensification of neural eCB signaling at CB1 receptors with repeated stress exposures.