The transcription factor Hex is expressed in the thyroid follicular cells (TFC) and in several other cell types. In TFC, Hex contributes to the control of the tissue-specific gene expression. By means of RT-PCR assays we found a correlation between the Hex and Pax8 (a different tissue-specific transcription factor, expressed in TFC) mRNA levels in normal and neoplastic thyroid tissues. This finding suggested the presence of a functional correlation between the two transcription factors. Therefore, we tested whether Pax8 regulates the transcriptional activity of Hex promoter. Indeed, by using cotransfection experiments in non-thyroidal cells, we show that increasing doses of Pax8 expression vector elicited a dose-dependent increase of the transcriptional activity of Hex promoter. Accordingly, gel-retardation assays indicated that in the Hex promoter are present several Pax8 binding sites. The Pax8 activating effect on Hex promoter was further increased by the contemporary presence of Hex protein. In fact, cotransfection of both Hex and Pax8 expression vectors doubled the transcriptional activity of Hex promoter with respect to the condition in which the Pax8 expression vector only was transfected. In addition, we show that also the transcriptional cofactor APE/Ref-1 cooperated with Pax8 for upregulation of Hex promoter activity. These findings, together with other published data, suggest that a network of functional interactions between transcriptional regulators is present in TFC.