2-[125I]iodomelatonin binds in vitro to murine mammary gland membranes, in a specific, saturable, reversible and temperature-dependent and pH-dependent way, displaying an affinity within the low nM range. The order of affinity to displace the radioligand from mammary membranes was: 6-hydroxy-melatonin > 5-hydroxyindoleamines > noradrenaline > 6-chloromelatonin > 5-methoxyindoles (melatonin included). There was a diurnal rhythm in Kd and Bmax of 2-[125I]-iodomelatonin binding, displaying the highest affinity and the lowest binding site concentration during the scotophase. The functional significance of melatonin binding sites was indicated by changes in binding parameters related to different rates of mammary growth during puberty, estrous cycle, pregnancy, lactation and the postlactation period.