2-Methoxyestradiol (2-ME2) is a natural estrogen metabolite that, while devoid of estrogenic effects, has both antiangiogenic and antitumor effects. 2-ME2 is currently being evaluated in Phase I and Phase II clinical trials for the treatment of multiple types of cancer. Novel analogues of 2-ME2 were tested for activities that predict antiangiogenic and antitumor effects. Selected analogues were tested for inhibitory activity against endothelial cell proliferation and invasion. The results show that these analogues are effective inhibitors of endothelial cell activities that may predict antiangiogenic activity, and one analogue, 2-methoxy-14-dehydroestradiol (14-dehydro-2-ME2), was 6-15-fold more potent than the parental compound in these assays. The analogues were also evaluated for inhibition of proliferation and cytotoxicity against multiple tumor cell lines and found to be potent and effective. 14-Dehydro-2-ME2 was approximately 15-fold more potent than 2-ME2 against various tumor cell lines, and 2-methoxy-15-dehydroestradiol was particularly effective against DU 145 and PC3 prostate cancer cell lines. In vivo antitumor activity was observed for the three analogues tested in the murine xenograft MDA-MB-435 model; however, 2-ME2 provided no antitumor activity in this trial. The two most effective analogues, 14-dehydro-2-ME2 and 2-methoxyestradiol-15 alpha,16 alpha-acetonide, provided 29.4% and 26.7% inhibition of tumor burden, respectively. Mechanism of action studies indicate that the analogues cause mitotic spindle disruption, mitotic arrest, microtubule depolymerization, and inhibition of the assembly of purified tubulin similar to the effects of 2-ME2. Consistent with antimitotics that inhibit the dynamic instability of tubulin and initiate apoptosis, these novel 2-ME2 analogues cause Bcl-2 phosphorylation and activation of mitogen-activated protein kinase signaling pathways.