Products of HOX genes are transcription factors responsible for developmental regulation and postnatal tissue homeostasis. Besides their well-established function played during embryonic development, we had previously demonstrated the direct role of HOXB7 in tumor progression through transactivation of several genes involved in the proliferative and angiogenic processes. This role is at first exerted through the deregulated, constitutive expression of this gene. To define the factors possibly responsible for such activation, we studied the molecular regulation of HOXB7 in embryonic and neoplastic cells. In a 1.9-kb 5' promoter region, we identified and functionally tested, at least in vitro, different regulatory sequences showing a direct binding by the NF-Y, YY1, Sp1/Sp3 and upstream stimulatory factor 1 (USF-1) transcription factors. Cell transfection and site-specific mutagenesis demonstrated Sp1/Sp3, NF-Y, YY1 and USF-1 binding to be functional and fundamental in driving HOXB7 expression. Disruption of the corresponding sites reduces gene expression of 65%, 78% and 55%, respectively. Because HOXB7 seems to play an important role in tumor proliferation and progression, the analysis of its regulatory sequences might represent an important step for gene targeting according to a new therapeutic strategy.