[125I]Iodomelatonin binding sites have been identified and characterized in kidneys of birds and mammals. These binding sites in the kidneys of guinea pig, duck and chicken were found to be stable, reversible, saturable, specific and of high affinity. The binding densities (Bmax) of [125I]iodomelatonin binding sites in the kidneys of guinea pig, duck and chicken ranged from 1.07 to 6.43 fmol/mg protein and the equilibrium dissociation constants (Kd) from 19.2 to 44.6 pmol/l at the middle of the light period (mid-light). It appears that [125I]iodomelatonin binding in the kidneys of mammalian species may have lower densities compared with birds. In the mammals studied, the guinea pig kidney showed the highest [125I]iodomelatonin binding. Pharmacological data indicated that the [125I]iodomelatonin binding to kidneys of guinea pig, duck and chicken was highly specific to melatonin, 2-iodomelatonin and 6-chloromelatonin. Diurnal variations in the Bmax of [125I]iodomelatonin binding sites were detected in the kidneys of duck and chicken with no difference in affinity. However, there was no diurnal variation in the Kd or Bmax in the guinea pig kidneys. The density of [125I]iodomelatonin binding sites in the cortex of guinea pig kidney was more than 8-fold higher than the binding in the medulla. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 10 mumol/l) reduced the Bmax and increased the Kd of [125I]iodomelatonin binding sites in the chicken kidney. However, in the membrane preparations of the guinea pig kidney, co-incubation with GTP gamma S (15 mumol/l) increased the Kd with no effect on the Bmax of the [125I]iodomelatonin binding. The effects of GTP gamma S on the kidney [125I]iodomelatonin binding suggest that these binding sites may couple to a G protein. The identification of [125I]iodomelatonin binding sites in the kidneys of mammals and birds supports the possibility of melatonin acting directly on the renal system.