A continuous flow perifusion system was used to measure the in vitro release rate, and percent release of endogenous norepinephrine (NE) and dopamine (DA) from rat brain fragments. Catecholamines (CA) were measured simultaneously in effluent fractions and in tissue fragments following perifusion using a modified radioenzymatic assay. Employing this perifusion system allows not only quantification of endogenous release, but also permits study of the dynamics of the release process. To validate the system, NE and DA release were measured in effluent from medial basal hypothalamus (MBH) and caudate nucleus (CN) fragments under various temperature and ionic conditions. K+-stimulated release (60 mM KCl), but not the basal release rate, is temperature and calcium dependent. Marked regional differences are seen in the parameters considered. DA release rate from the CN in male rats was 30 times greater than from the MBH. The percent of DA release was 2.5 times greater and there was a 3 times greater final tissue concentration of DA in the CN than in the MBH. Although the release rate of NE from CN fragments was higher than that of the MBH, it did not reach statistical difference. However, the percent of NE release from the CN was close to 4 times greater than the percent release from the MBH, and final tissue concentrations of NE were 87.9 pg/mg in the CN compared to 1,240 pg/mg in the MBH (postperifusion values). In a group of female rats, only the percent release of NE from the MBH was significantly different from that of similar tissue from male rats. However, the release rate and percent release of DA from the MBH of female rats were significantly lower than that of male rats. In appears that under this in vitro condition there are marked regional and sex differences in the metabolism of CA between the CN and the MBH fragments of male and female rats. Implications of these findings are briefly discussed.