Ketoconazole has been recently used in the primary treatment of patients with metastatic cancer of the prostate and is identified as a potent inhibitor of cytochrome P450-dependent adrenal and testicular androgen production. The drug has also shown activity in patients failing conventional hormonal manipulation. We subsequently showed that ketoconazole in vitro has a direct cytotoxic effect on human androgen-independent prostatic cancer cell lines. In order to better define the possible role of ketoconazole on hormone-independent prostatic cancer, we incubated the cells from human androgen-independent prostatic cancer lines in a methylcellulose tumour colony assay with different doses of the drug and increasing doses of conventional cytotoxic agents (etoposide, bleomycin, vinblastine, methotrexate, and teniposide). We demonstrated synergistic suppression of prostate cancer clonogenic cell growth by ketoconazole in the presence of vinblastine or etoposide. This observation may assign a new and important role for ketoconazole as part of combination chemotherapy in the treatment of patients with advanced prostatic cancer.