Hormone-refractory prostate cancer continues to be associated with a very poor prognosis. Agents that inhibit microtubule function have been found to be cytotoxic to prostate cancer cells in preclinical and clinical settings. It was the aim of this study to assess the activity of estramustine and colchicine, two microtubule inhibitors, in hormone-refractory prostate cancer. In clinically achievable concentrations, the combination of estramustine and colchicine was cytotoxic to both the Dunning rat prostate adenocarcinoma cell line MAT-LyLu (MLL) and human prostate cancer cells (PC-3). Microtubule function was assessed in vitro to evaluate possible mechanisms of action. In motility and cell cycle analysis assays, estramustine and colchicine inhibited cellular motility but not cell cycle transit. In vivo, these two agents both inhibited the growth of implanted Dunning rat prostate adenocarcinoma MLL cells but did not appear to have additive effects. The use of oral colchicine in the treatment of hormone-refractory prostate cancer requires further investigation.