High-affinity [ 3H]imipramine binding sites detected in the brain are associated with the neural uptake mechanism for serotonin. In this study [ 3H]imipramine binding in the retinal synaptosomal P 2-fraction, which contains the serotonin-accumulating nerve terminals, was characterized using two different animal species, chicken and pig, and with a wide range of imipramine concentrations. The specific binding of imipramine was saturable. When homogenates were prepared from fresh retinas, a high-affinity binding site in the nanomolar range and a low-affinity binding site in the micromolar range were always found. In the pig retina only a high-affinity binding site was found after freezing. The B max-value of the high-affinity binding was about 10 times greater in the retinas of pigs than in those of chickens; B max-values for fresh homogenates were 1711- and 235 fmol mg −1 protein and those for frozen homogenates were 2066- and 146 fmol mg −1 protein in pig and chicken retinas, respectively. Our results confirm the complexity of imipramine binding described for different regions of the brain. The great difference in the B max-values of the high-affinity binding in chicken and pig retinas might indicate differences between species in the serotonergic system.