The DNA sequences corresponding to a DNaseI-hypersensitive region identified previously in bovine thyroglobulin gene chromatin (Hansen et al. (1988) Eur. J. Biochem. 178, 387-393) exhibited the properties of a transcriptional enhancer in a transient assay in primary cultured dog thyrocytes, but did not so in transfected HeLa cells. By contrast to the thyroglobulin proximal promoter, the enhancer element did not require cyclic AMP stimulation of the thyrocytes to be active. Using a bi-directional deletion approach, the minimal region displaying enhancer activity has been localized between positions -1906 and -1744 relative to the thyroglobulin gene transcription start. DNA-footprinting experiments revealed the presence of several binding sites for the thyroid-specific transcription factor TTF-1 within the enhancer sequence.