Op18 (Oncoprotein 18, Stathmin) is a mitotic regulator that is highly expressed in many cancers. We have characterized four functional CCAAT boxes in the Op18 gene located at positions: -980, -745, -599 and -65, relative to the transcriptional start site. NF-Y is a ubiquitously expressed CCAAT-binding transcription factor that regulates a number of cell cycle controlled genes. We have used promoter-reporter assays and mobility shift assays to functionally examine these CCAAT boxes. All sites contribute to the basal expression of Op18, with the sites at -980 and -599 being repressive and the sites at -745 and -65 being stimulatory. Mobility shift assays indicate that all CCAAT box sites bind factors in nuclear extracts from Hek293. However, only the repressive site at -599 and the stimulatory site at -65 are competent to bind NF-Y, suggesting that NF-Y may play a role in promoting both activation and repression of Op18 expression. The NF-Y site at -65 accounts for greater than 60% of the Op18 gene expression. EMSA competition studies indicate that NF-Y binds with a much higher affinity to the -65 site than to the -599 site, suggesting that in asynchronously growing cells NF-Y functions only to stimulate expression through the -65 binding site. These data suggest that NF-Y is a major transcription factor promoting expression of Op18.