The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodomain (TTF- 1HD) has been investigated. Methylation and ethylation interference experiments show that the TTF-1HO alone recapitulates the DNA binding properties of the entire protein. Studies carried out with mutant derivatives of TTF-1HD indicate a precise correspondence of some of its amino acid residues with specific bases in its binding site, allowing a crude orientation of the TTF- 1HD within the protein - DNA complex. TTF-1HD shows an overall geometry of interaction with DNA similar to that previously observed for Antennapedia class HDs, even though the binding specificities of these two types of HDs are distinct. We demonstrate that the crucial difference between the binding sites of Antennapedia class and TTF-1 HDs is in the motifs 5′-TAAT-3′, recognized by Antennapedia, and 5′-CAAG-3′, preferentially bound by TTF-1. Furthermore, the binding of wild type and mutants TTF-1 HD to oligonucleotides containing either 5′-TAAT-3′ or 5′-CAAG-3′ indicate that only in the presence of the latter motif the Gln50 in TTF-1 HD is utilized for DNA recognition. Since the Gin at position 50 is an essential determinant for DNA binding specificity for several other HDs that bind to 5′-TAAT-3′ containing sequences, we suggest that utilization by different HDs of key residues may depend on the sequence context and probably follows a precise hierarchy of contacts.