Previous clinical trials have suggested that hydroxyurea may possess some activity against prostate cancer. The in vitro antiproliferative activity of hydroxyurea was evaluated in three hormone-refractory prostate cancer cell lines, PC-3, DU-145 and PC-3M. Fifty-percent inhibition of growth in all three cell lines required prolonged (120 h) exposure to hydroxyurea at a concentration of approximately 100 microM. Using pharmacokinetic data obtained during the course of a clinical trial of hydroxyurea, we simulated a dosing regimen that would sustain plasma drug concentrations above 100 microM for 120 h (1 g loading dose, followed by 500 mg every 6 h for 5 days in a 70 kg man). Since this dosing regimen is likely to generate an unacceptable degree of myelosuppression, in vitro combination studies were conducted with hydroxyurea and phenylbutyrate, a new differentiating agent with no myelosuppressive effects. These studies resulted in a reduction of the hydroxyurea concentration necessary for 50% growth inhibition (50 microM of hydroxyurea plus 0.5 mM of phenylbutyrate). A regimen designed to achieve that hydroxyurea concentration (400 mg loading dose, followed by 200 mg every 6 h for 5 days) should be clinically achievable. Based on these results, this combination deserves further evaluation in patients with stage D prostate cancer.