It has recently been demonstrated that L-365,260, a CCK-B antagonist in mammals, causes an increase in food intake in chickens. In contrast, L-364, 718, a CCK-A antagonist in mammals, shows this effect only at very high dose levels. It has been shown that L-365,260 has very low affinity for chicken CCK receptors. Thus, the mechanism of action of L-365,260 remains unknown. As L-365,260 is a benzodiazepine derivative, one may hypothesize that it would be acting on benzodiazepine binding sites. The aims of this work were to establish the existence of benzodiazepine binding sites in the chicken brain, and to check the possibility that L-365,260 was acting on these receptors, determining the affinity of L-364,718 and L-365,260 for them. We have found specific binding for tritiated flunitrazepam (a benzodiazepine agonist) ([3H]-flunitrazepam) in chicken brain membranes. A single binding site was detected with a Kd of 3.58 +/- 0.97 nM and a Bmax of 451.6 +/- 23.3 fmol/mg protein L-365,260 and L-364,718 exhibited very low affinity for these binding sites (Ki = 1.17 x 10(-6) +/- 0.16 x 10(-6) M and Ki > 10(-5) M, respectively). Thus, these results demonstrate that the increase in food intake caused by L-365,260 in the chicken is not due to a direct action on benzodiazepine receptors. Other possible explanations for its effect are discussed.