The time-course of response to hypothalamic CRF was determined and used to design a CRF assay in which lesioned rats could be employed several times within the same day. Hypothalamic extract over the dose-response range was administered to rats bearing hypothalamic lesions and the resultant corticosterone elevations were monitored at specified intervals thereafter. Corticosterone responses after 0.5, 1, or 2 hypothalamic (ME) equivalents were examined at 20, 40, 60, and 120 min after intravenous injection. Each dose produced a peak elevation at 20 min, followed by a return to baseline levels over a 2 h period. After 0.5 ME, baseline corticosterone levels were observed by 40 min; after 1 ME, a return to baseline values was noted at 60 min; whereas after a maximal dose of 2 ME, corticosterone levels approached the baseline values at 120 min. These results suggested that lesioned rats might be used at 2 h intervals for CRF assay. Responsiveness and specificity were evaluated after multiple use of lesioned rats for CRF assay. The responses to ME-extract or to control injections were consistent and specific, irrespective of the injection sequence. Lesioned animals were used 4 times at 2 h intervals, thereby providing for an improved, more practical and reliable CRF assay.