An 18-bp enhancer controls cell-specific expression of the calcitonin/calcitonin gene-related peptide (CT/CGRP) gene. The enhancer is bound by a heterodimer of the bHLH-Zip protein USF-1 and -2 and a cell-specific factor from thyroid C cell lines. In this report we have identified the cell-specific factor as the forkhead protein Foxa2 (previously HNF-3ß). Binding of Foxa2 to the 18-bp enhancer was demonstrated using electrophoretic mobility shift assays. The cell-specific DNA-protein complex was selectively competed by a series of Foxa2 DNA binding sites and addition of Foxa2 antiserum supershifted the complex. Likewise, a complex similar to that seen with extracts from thyroid C cell lines was generated using an extract from heterologous cells expressing recombinant Foxa2. Interestingly, overexpression of Foxa2 activated the 18-bp enhancer in heterologous cells, but only in the presence of the adjacent HLH motif. Likewise, coexpression of USF proteins with Foxa2 yielded greater activation than by Foxa2 alone. Unexpectedly, Foxa2 overexpression repressed activity in the CA77 thyroid C cell line, suggesting that Foxa2 may interact with additional cofactors. The stimulatory role of Foxa2 at the CT/CGRP enhancer was confirmed by siRNA-mediated knockdown of Foxa2. As seen with Foxa2 overexpression, the effect of Foxa2 knockdown also required the adjacent HLH motif. These results provide the first evidence for combinatorial control of gene expression by bHLH-Zip and forkhead proteins.