In the present work we have characterized the long-term influence of a single exposure to the stress of immobilization (IMO) on the hypothalamic–pituitary–adrenal (HPA) axis of adult rats. Rats without prior stress (control) and rats exposed to IMO for 2 h on day 1 (IMO+4wk) or on day 21 (IMO+1wk) were killed on day 28, either without stress (basal), immediately after IMO for 1 h (IMO), or 1 h after termination of IMO (post-IMO). IMO caused a strong activation of c-fos mRNA and corticotropin-releasing factor (CRF) and vasopressin (AVP) heteronuclear RNA (hnRNA) in the paraventricular nucleus of the hypothalamus in control rats; this activation was essentially maintained in the post-IMO period. The overall AVP hnRNA response to day 28 stress was not affected by prior stress. Post-IMO c-fos mRNA and CRF hnRNA levels were lower in previously stressed rats, as compared with controls. Whereas the effect of prior IMO on both peripheral HPA hormones and c-fos mRNA was maximal in IMO+1wk rats, the effect of prior stress on CRF hnRNA was only observed in IMO+4wk rats. The present data indicate that prior single IMO triggers a process of desensitization of the HPA responsiveness to IMO over the course of the following weeks. Although the various components of the HPA axis were modified in the same direction, a clear temporal dissociation was found among them, revealing the fine tuning of stress-induced activation of the HPA axis.