The mammary gland factor MGF has been described as a developmentally and environmentally regulated nuclear factor required for transcription of the milk protein gene beta-casein. In the current study the individual role of lactogenic hormones in the activation of MGF DNA binding and the functional relation of MGF to known transcription factors was investigated by electrophoretic mobility shift assays. DNA binding of MGF was rapidly induced by PRL in mammary epithelial cells. The activation was not inhibited by the protein synthesis inhibitor cycloheximide. The effect of PRL on MGF did not require costimulation of cells with the other lactogenic hormones, insulin, and glucocorticoids. Thus, MGF is the first example of a nuclear factor directly regulated by PRL. The MGF complexes formed upon initiation of lactation in the mammary gland and upon stimulation of mammary epithelial cells with PRL migrated at the same position in electrophoretic mobility shift assay, whereas the MGF complex found in mammary gland extracts of pregnant mice exhibited a faster mobility. In cell cultures, PRL-induced activation of MGF as well as up-regulation of beta-casein gene transcription was confined to confluent cultures of mammary epithelial cells and inhibited by long term incubation of cells with epidermal growth factor. MGF was found to be related to the nuclear factors that are activated by tyrosine phosphorylation when cells are stimulated with interferons or cytokines. This notion is supported by experimental evidence for phosphorylation of MGF on tyrosine and by the similar DNA recognition motifs of MGF and cytokine-activated factors.