Melatonin receptors were studied in quail livers using the melatonin agonist 2-[125I]iodomelatonin ([125I]MEL) as the radioligand. The specific binding of [125I]MEL to membrane preparations of liver was rapid, stable, saturable, reversible and of high affinity. Scatchard analysis of the specific binding data indicated an equilibrium dissociation constant (Kd) of 19.4 +/- 1.01 pmol/l (n = 7) and a maximum number of binding sites (Bmax) of 1.16 +/- 0.19 fmol/mg protein (n = 7) in the quail liver collected at mid-light. The Hill coefficient approached 1.0, suggesting a single class of [125I]MEL binding sites in the quail liver. The diurnal variation study showed that the value of Kd was 64.4% higher (p < 0.05) at mid-dark compared to mid-light, with no significant change in Bmax. The kinetic analysis showed that the Kd value was 25.0 +/- 3.94 pmol/l at mid-light, which was comparable with values determined from saturation studies. Aside from 2-iodomelatonin, melatonin and 6-chloromelatonin, all indole analogs and neurotransmitters tested in inhibition studies had slight or no displacement of [125I]MEL binding. These studies demonstrated that [125I]MEL binding sites were highly specific for melatonin. The presence of 10 and 50 mumol/l guanosine 5'-O-(3-thiotriphosphate) significantly increased (p < 0.05) the Kd values and depressed the Bmax values, proposing that [125I]MEL binding sites in quail livers were coupled to a G-protein. Our results indicate that melatonin may exert a direct action on liver functions.