We have used a gel retardation assay to follow the purification of a calf thyroid nuclear protein that binds to the -70 region of the rat thyroglobulin promoter. The activity producing the observed band shift is thyroid specific. The same shift is in fact observed with extracts prepared from a differentiated rat thyroid cell line which synthesizes and secretes thyroglobulin, while no similar shift is detected when cell unable to express their endogenous thyroglobulin gene or tissues different from thyroid are used as a source of nuclear extract. Competition experiments suggest that the same protein may bind at two different sites within the promoter. The two sites display considerable sequence homology. Sequence comparisons between the rat, calf and human promoter suggest that more than the sequence is the geometry of the promoter which is conserved.