Corticotropin-releasing hormone (CRH) gene transcription and mRNA expression are keyed to stimuli activating the pituitary-adrenocortical axis, suggesting a connection between neuronal activation and synthesis of active peptide. However, the relationship between CRH mRNA and levels of CRH peptide remains to be definitively established. The present report characterizes an immunoautoradiographic (IAR) strategy to assess CRH peptide expression in an anatomical context. Non-fixed tissue sections through the rat hypothalamus were reacted with a primary antibody against rat CRH, followed by incubation with [35S] or [125I] labeled secondary antibody. Autoradiography performed on reacted sections revealed that CRH immunoreactivity could be detected in CRH-containing regions of the hypothalamus and amygdala. Generation of CRH signal was blocked by preabsorption of primary antibody with CRH peptide, demonstrating antibody specificity. IAR performed on nitrocellulose blotted with synthetic CRH peptide revealed a linear relationship between peptide quantity and intensity of autoradiographic signal, verifying that this method is appropriate for semi-quantitative analysis of CRH peptide regulation. Assessment of CRH peptide regulation revealed a significant increase in CRH content in adrenalectomized rats (ADX) relative to sham-adrenalectomized (SHAM) controls (196%). In situ hybridization performed on adjacent sections revealed a similar increase in CRH mRNA expression in ADX rats (256%), and a significant correlation between CRH peptide and mRNA measures (r = 0.68). No ADX induced changes were seen in median eminence, dorsomedial hypothalamus or central amygdaloid nucleus. The results of this study indicate that CRH biosynthesis appears to be driven by amount of available mRNA, rather than changes in translational efficacy. In addition, the IAR technique appears ideally suited to allow concomitant assessment of mRNA and protein expression within defined populations of CNS neurons.