The chromatin structure of the growth hormone gene and flanking DNA was analyzed in GC rat pituitary tumor cells, which appropriately express and regulate the gene. Thyroid hormone induced the DNase I hypersensitivity of a chromatin domain spanning the transcriptional initiation site from approximately -200 to +150. Three inducible hypersensitive sites were discerned in this region. One of these sites was in the first intron of the gene. The second site was within a region of 5' flanking DNA which promotes accurate, thyroid hormone-regulated transcriptional initiation. Centered between these two sites was a third hormone-inducible hypersensitive site mapping at the position of the TATA sequence. The hormone responsiveness of these hypersensitive sites suggests that occupied 3,5,3'-triiodo-L-thyronine receptor interacts with sequences flanking the growth hormone promoter, perhaps facilitating the binding or activation of a transcription factor at the TATA homology. Three hormone-independent hypersensitive sites were identified near or within intron or flanking DNA sequences similar to Chinese hamster ovary type 2 repetitive DNA. Two additional hormone-independent hypersensitive sites were located more than 1000 nucleotides up-stream of the growth hormone promoter. These sites were present in the cloned genomic DNA and were the only distinct sites found in rat spleen and cultured rat liver cells. Thyroid hormone treatment of GC cells appeared to increase the moderate DNase I sensitivity of the growth hormone gene region beyond that found in deinduced or glucocorticoid-treated cells. Dexamethasone had no discernible effect on the chromatin structure of the gene or flanking DNA in these studies.