The subfornical organ (SFO) is an important sensory circumventricular organ implicated in the regulation of fluid homeostasis and energy balance. We investigated whether the SFO is activated by the hormone cholecystokinin (CCK). CCK₁ and CCK₂ receptors were identified in the SFO by RT-PCR. Dissociated SFO neurons that responded to CCK (40/77), were mostly depolarized (9.2±0.9mV, 30/77); but some were hyperpolarized (-7.3±1.1 mV, 10/77). We next examined the responses of SFO neurons in vivo to CCK (16µg/kg, i.p.), in the presence and absence of CCK1 or CCK2 receptor antagonists (Devazepide; 600 µg/kg and L-365,260; 100 µg/kg, respectively), using the functional activation markers c-Fos and p-ERK. The nucleus of the solitary tract (NTS) served as a control for CCK-induced activity. There was a significant increase in c-Fos expression in the NTS (259.2±20.8 neurons) compared to vehicle (47.5±2.5). Similarly, in the SFO, c-Fos was expressed in 40.5±10.6 neurons in CCK-treated compared to 6.6 ±2.7 in vehicle-treated rats (P<0.01). Devazepide significantly reduced the effects of CCK in the NTS, but not in SFO. L-365,260 blocked the effects of CCK in both brain regions. CCK increased the number of p-ERK neurons in NTS (27.0±4.0) as well as SFO (18.0±4.0), compared to vehicle (8.0 ±2.6 and 4.3±0.6, respectively; P<0.05). Both devazepide and L-365,260 reduced CCK induced p-ERK in NTS, but only L-365,260 reduced it in the SFO. In conclusion, the SFO represents a novel brain region at which circulating CCK may act via CCK₂ receptors to influence central autonomic control.