Developmental stage- and tissue-specific expression of the rat growth hormone (rGH) gene is conferred by DNA sequences within 237 base pairs of the transcription start site. Although binding of a number of transcription factors including Pit-1, Sp1, GHF3, and thyroid hormone receptor (T3R) stimulates rGH expression, several studies have suggested that interactions between these factors are important in determining cell specificity and responsiveness to extracellular signals. We have directly tested this hypothesis by creating a set of nested insertional mutations at two positions in the rGH promoter. Sequences were inserted at either position -148, separating GHF-3 and T3R binding sites from the downstream Pit-1 and Sp 1 binding sites, or at -51, separating the above elements from the TATA box. All insertions were made in the context of the rGH gene -237/+8 5'-flanking DNA, linked to a chloramphenicol acetyltransferase reporter gene and tested for activity by transient transfection in GC pituitary tumor cells. Insertions at both -148 and -51 caused sharp distance-dependent reductions in serum-stimulated expression such that insertions of 23 base pairs at -51 or 44 base pairs at -148 were sufficient to isolate the effects of sequences upstream of the insertion point. Insertions at -148 reduced T3 responsiveness severalfold but had little or no effect on stimulation by forskolin, whereas insertions at -51 reduced both T3 and forskolin responsiveness. Our results are consistent with the hypothesis that expression and regulation of the rGH gene is dependent on short-range protein-protein interactions, which are more critically dependent on spacing than the relative orientation of the transcription factor binding sites.