Functional analysis of remote 5'-flanking sequences from the human thyroglobulin gene in primary cultured dog thyrocytes led to the identification of a partly cAMP-responsive enhancer, located between -3.6 to -2.2 kb from the transcriptional start site. Deletion analysis of the 1.4 kb-long region localised the enhancer activity in a 0.5 kb-long fragment (located between -3.2 and -2.7 kb relative to transcription start), which could be divided into two functional sub-fragments of 0.2 and 0.3 kb. A potential binding site for the CREB/ATF transcription factors was found in the 0.3 kb element. The complete enhancer region had no detectable activity when assayed in Hela cells, suggesting that it constituted a thyroid-specific regulatory element. Accordingly, footprinting experiments revealed the presence of several binding sites for Thyroid Transcription Factor-1 (TTF-1) in both the 0.2 and 0.3 kb elements.