A new set of estradiol derivatives bearing various substituents at the 2-position were synthesized in order to further elucidate the structural parameters associated with the antitubulin activity and cytotoxicity of 2-substituted estradiols. The potencies of the new compounds as inhibitors of tubulin polymerization were determined, and the cytotoxicities of the analogues in human cancer cell cultures were investigated. The substituents introduced into the 2-position of estradiol included E-3'-hydroxy-1'-propenyl, 2'-hydroxyethoxy, 3-N,N-dimethylaminoethylideneamino, 2'-hydroxyethylineneamino, (beta-3,4,5-trimethoxyphenyl)ethenyl, phenylethynyl, ethynly, 1'-propynyl, and cyano. The substituents conferring the ability to inhibit tubulin polymerization included E-3'-hydroxy-1'-propenyl, 2'-hydroxyethoxy, ethynyl, and 1'-propynyl. The remaining compounds were all inactive as inhibitors of tubulin polymerization when tested at concentrations of up to 40 microM. All of the compounds were cytotoxic in a panel of 55 human cancer cell cultures, and in general, the most cytotoxic compounds were also the most potent as inhibitors of tubulin polymerization. 2-(1'-Propynyl)estradiol displayed significant anticancer activity in the in vivo hollow fiber animal model.