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Interactions between DNA sequences and nuclear proteins are important in the regulation of gene expression. We have applied a gel retardation method to examine the binding of nuclear proteins from the rat pituitary gland to regulatory sequences of the rat TSH β-subunit gene. Binding between nuclear protein and DNA is demonstrated by an alteration in electrophoretic mobility of the DNA. A 0·4 m NaCl fraction of pituitary nuclear proteins bound to a fragment of DNA containing the promoter region of the TSH-β gene, but not to plasmid DNA or insulin cDNA of comparable size. This nuclear protein fraction also bound to fragments of DNA containing sequences from the 5'-flanking region of the gene; binding of nuclear proteins was evident as far as 1 kb 5' to the structural gene. Each of these regions of the TSH-β gene formed a number of distinct complexes with the nuclear protein extract, distinguished by their differing binding affinities in the presence of poly(dI-dC), and by their electrophoretic mobility. These findings suggest that transcriptional regulation of the rat TSH-β gene may be mediated by interaction of a number of nuclear proteins with regulatory sequences of DNA up to 1 kb upstream of the transcriptional start site of the gene.