Thyroid dysgenesis is the most frequent cause of congenital hypothyroidism, but its molecular pathophysiology is largely unknown. Our hypothesis that some genes downstream to thyroid transcription factor-2 (TTF-2) might be responsible for development of the thyroid prompted us to identify genes whose expression is stimulated by TTF-2. PCR products of cDNA clones obtained by a subtraction PCR method in TTF-2 expressing cell lines were screened with labeled cDNA by microarray analysis. We isolated 17 genes up-regulated by TTF-2, which were subsequently confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). One of them is a novel gene designated T1560 that showed a highly thyroid-specific expression pattern. Luciferase reporter assays showed that expression of all of the 14 genes tested was stimulated by both TTF-2 and TTF-1, another thyroid-specific transcription factor. Our results have important implications for understanding normal thyroid development as well as the molecular defects underlying thyroid dysgenesis.