Abstract An in vitro autoradiographic assay was used in identifying a magnesium-dependent, non-specific binding of [ 125I] prolactin to myelinated fiber tracts in the rat brain. Frozen tissue sections were incubated for 18 h at 4°C in media which included [ 125I] prolactin alone or with a 500 fold excess of unlabelled prolactin. Magnesium in the incubation medium caused a non-specific binding of radiolabelled prolactin to the myelinated fiber tracts in the brain. In contrast, calcium did not facilitate prolactin non-specific binding to myelin. Hence, calcium should optimize the detection of specific prolactin binding sites in the brain by in vitro autoradiographic or radioreceptor assays.