The phosphorylation of thyroid transcription factor-1 (TTF-1), is homeodomain-containing transcription factor that is required for thyroid-specific expression of the thyroglobulin and thyroperoxidase gene promoters, has been studied. Phosphorylation occurs on a maximum of seven serine residues that are distributed in three tryptic peptides. Mutant derivatives of TTF-1, with alanine sites, have been constructed and used to assess the functional relevance of TTF-1 phosphorylation. The DNA binding activity of TTF-1 appears to be phosphorylation-independent, as indicated also by the performance of TTF-1 purified from an overexpressing Escherichia coli strain. Transcriptional activation by TTF-1 could require phosphorylation only in specific cell types since in a co-transfection assay in heterologous cells both wild-type and mutant proteins show a similar transcriptional activity.