We aimed to support in vitro the glucosensing capacity observed in vivo in rainbow trout hypothalamus, hindbrain, and Brockmann bodies (BB), and to obtain preliminary evidence of the mechanisms involved. The response of parameters involved in glucosensing capacity (HK, GK and PK activities, and glucose and glycogen levels) was assessed in these tissues incubated for 1 h with 2, 4 or 8 mM D-glucose alone (control) or with specific agonists/inhibitors of the steps involved in glucosensing capacity in mammals. These agents were a competitor for glucose phosphorylation (15 mM mannose), SUR-1 effectors (500 µM tolbutamide or diazoxide), glycolytic intermediates (15 mM glycerol, lactate or pyruvate), and inhibitors of glucose transport (10 µM cytochalasin B), glycolysis (20 mM 2-DG) and L-type calcium channel (1 µM nifedipine). Control incubations of the three tissues displayed increased glucose and glycogen levels and GK activities in response to increased medium glucose thus supporting our previous in vivo studies. Furthermore, critical components of the glucosensing mammalian machinery are apparently functioning in the three tissues. The responses in brain regions to all substances tested (except 2-DG and nifedipine) were similar to those observed in mammals suggesting a similar glucosensing machinery. In contrast, in BB only the effects of 2-DG, lactate, pyruvate, diazoxide and nifedipine were similar to those of mammalian -cells, suggesting that some of the components of the piscine glucosensing model are different than those of mammals. Such differences may relate to the importance of amino acids rather than glucose signalling in the trout BB.