The natural estrogen metabolite 2-methoxyestradiol (2ME) is anti-angiogenic in vivo and a strong growth inhibitor in vitro. The growth inhibition is due to mitotic arrest and apoptosis. These effects are reminiscent of those induced by taxol, and appear to be mediated by inhibition of microtubule dynamics. Here we have studied the cellular response to 2ME in regard to potential mediators of the observed cellular changes. 2ME treatment increases the insoluble polymerized fraction of cellular tubulin similar to taxol, and in contrast to the microtubule depolymerizing drugs such as colcemid and vincristine. This stabilization following 2ME treatment is accompanied by phosphorylation and inactivation of Bcl-2 increasing gradually from 2-24 hours. To study the pathway leading to Bcl-2 phosphorylation we analyzed Raf-1 and JNK/SAPK kinases, both of which have been reported to be involved in Bcl-2 inactivation. Our results indicate that Raf-1 is phosphorylated in response to 2ME, but this occurs later than Bcl-2 phosphorylation suggesting that Raf-1 is not directly phosphorylating Bcl-2. JNK/SAPK was activated rapidly after 2ME treatment. However, this activation was transient and returned to undetectable levels by 2 hours of treatment, demonstrating that JNK/SAPK is not directly phosphorylating Bcl-2. Taken together with previous results indicating that overexpression of JNK/SAPK leads to Bcl-2 phosphorylation, our results would support a model where JNK/SAPK is indirectly phosphorylating Bcl-2.