Op18 (Oncoprotein 18, Stathmin) is an important mitotic regulator that is highly expressed in many cancers including the metastatic prostate carcinoma cell line PC-3-M. Recent studies indicate that antisense-mediated down-regulation of Op18 can inhibit cellular proliferation. However, the transcriptional mechanisms responsible for its normal regulation and for its high level of expression in proliferating cells remain poorly understood. In the study presented here, we have characterized transcription factor binding sites that together account for nearly 80% of the Op18 expression in PC-3-M cells. The 5' flanking region of the Op18 gene contains four putative E2F sites located at -700 (site 1), -28 (site 2), -19 (site 3), and +720 (site 4) relative to the transcriptional start site. E2F has been implicated in both the c-Jun-mediated up-regulation and the doxorubicin-induced repression of Op18 expression. We have used promoter-reporter assays and mobility shift assays to functionally examine each of these E2F sites. Mutagenesis studies indicate that all sites contribute to the basal expression of Op18. Mutagenesis of either site 1 or 4 reduced the reporter activity by 40%, mutagenesis of site 2 reduced reporter activity by 20%, and mutations in site 3 had no effect on reporter activity. Combinatorial mutagenesis indicates that site 1 and 4 function independently, whereas site 2 functions synergistically with either site 3 or 4. Mobility shift assays indicate that all elements bind factors in the nuclear extracts of PC-3-M cells. Characterization of the sites show that site 1, though a positive element, is not E2F specific; sites 2 and 3 may contain an overlapping binding site for E2F and NF1; and site 4, which resides in intron 1, is the only site shown to interact exclusively with E2F. These studies suggest that the overexpression of Op18 in PC-3-M cells is mediated predominantly through the E2F family of transcription factors.