We have previously shown that DNA elements controlling tissue specific expression of the rat thyroglobulin gene extend 170 bp upstream of the cap site and have identified a thyroid specific nuclear factor which binds the promoter in the -60 region (site C). Here we report that the distal portion of the promoter, extending from -160 to -120, contains two contiguous DNA elements (sites A and B) which interact with the same thyroid-specific factor binding to proximal site C. A second nuclear factor, ubiquitously distributed, binds to the distal site A. Transient cotransfection-competition studies show that all the three binding sites A, B and C titrate a trans-acting factor(s) which is necessary for the transcription of the thyroglobulin gene.