The segment polarity gene Fused (Fu) encodes a putative serine-threonine kinase Fu, which has been shown to play a key role in the Hedgehog signaling pathway of Drosophila. Human FU (hFU) has been shown to enhance the activity of Gli transcription factors, targets of the signaling pathway. However, Fu ( -/- ) mice do not show aberrant embryonic development indicating that mouse Fu (mFu) is dispensable for Hedgehog signaling until birth. In order to investigate if there are important differences between hFU and mFu, we cloned the cDNA, analyzed expression and tested the ability of mFu to regulate Gli proteins. Of the tested tissues only brain and testis showed significant expression. However, in transient overexpression analyses mFu was able to enhance Gli induced transcription in a manner similar to hFU. Thus, we turned to RNAi in order to test if mFu would be important for Hedgehog signaling after all. In one cell line with reduced mFu expression the Hedgehog signaling was severely hampered, indicating that mFu may have a role in Hedgehog signaling and Gli regulation in some cellular situations.