The dorsomedial hypothalamus (DMH) has been implicated in the coordination of stress responses. Restraint stress or systemic corticosterone (CORT) treatment induces a rapid increase in tissue concentrations of serotonin (5-hydroxytryptamine; 5-HT) in the DMH. Although the mechanism for rapid changes in 5-HT concentrations in the DMH is not clear, earlier results suggest that stress-induced increases in CORT may inhibit 5-HT transport from the extracellular fluid by acting on corticosterone-sensitive organic cation transporters (OCTs). We tested the hypothesis that perfusion of the medial hypothalamus (MH), which includes the DMH, with the OCT blocker decynium 22 (D-22) would potentiate the effects of mild restraint on extracellular 5-HT. Male Sprague-Dawley rats, implanted with a microdialysis probe into the MH, were treated with reverse-dialysis of D-22 (20 microM; 40 min) or vehicle and subjected to either 40 min mild restraint or undisturbed control conditions. Perfusates collected from a separate group of rats were evaluated for the effect of restraint on extracellular CORT concentrations in the MH. Reverse-dialysis of D-22 induced an increase (200%) in extracellular 5-HT concentrations in the MH in undisturbed control rats. Restraint in the absence of D-22 did not significantly affect MH CORT or 5-HT concentrations. However, perfusion of the MH with D-22 during restraint led to an increased magnitude and duration of extracellular 5-HT concentrations, relative to D-22 by itself. These results are consistent with the hypothesis that OCTs in the DMH contribute to the clearance of 5-HT from the extracellular fluid under both baseline conditions and mild restraint.