Whereas most eukaryotic sequence-specific DNA binding proteins exclusively recognize native double-strand sequences, there is increasing evidence that sequence-specific, single-strand DNA-binding proteins (SSBP) are involved in the regulation of gene expression. The GHR is essential for the actions of GH on growth and metabolism. Electromobility shift assays (EMSA) established that a 42-bp enhancer element in the promoter of the pregnancy-specific L1 transcript of the GHR bound nuclear proteins specific for the coding strand or the DNA-duplex. Using methylation interference footprinting and EMSA with mutant oligonucleotides, the DNA binding sites for the SSBP and the double-strand DNA-binding protein (DSBP) were mapped and shown to be contigous with partial overlap. Shift-Western analysis indicated that the SSBP was a component of the DSBP complex. A functional interaction between SSBP and DSBP was suggested by the effect of the exclusion of binding of SSBP on equilibrium binding and dissociation rate (off rate) of the DSBP-DNA complex. Experiments using the anionic detergent deoxycholate provided evidence for a direct protein-protein interaction between SSBP and DSBP. Using lectin-affinity chromatography, discordance between the pattern of O-glycosylation of SSBP and DSBP was demonstrated. Transient transfection experiments support the role of SSBP as a repressor of DSBP's activation of transcription of the GHR gene. South-Western analysis indicated that a protein of molecular weight 23-kd exhibited binding activity specific to the coding strand of the enhancer element. We conclude that single- and double-strand DNA binding proteins conjointly regulate expression of the GHR gene. The GHR gene joins a family of biologically relevant genes whose expression is modulated by SSBPs.