Bisphosphonates (BPs), specific inhibitors of osteoclasts, are well established in the management of skeletal metastases in breast cancer. Recent studies have suggested that these compounds may also directly influence tumor cell proliferation. As adrenocortical cancer frequently leads to bone metastases, we investigated the effects of clodronate (CLO) in the human adrenocortical cancer cell line NCI-H295 and in primary cultures of bovine adrenocortical cells. Both the non-amino BP CLO and the amino BP pamidronate (PAM) exhibited a dose-dependent antiproliferative effect in both cell types (cell viability in NCI-H295 cells: 100 microM CLO: 98+/-8%; 500 microM CLO 76+/-6%; 1000 microM CLO 53+/-2; 10 microM PAM 96+/-3%; 50 microM PAM 46+/-6%; 100 microM PAM 11+/-1% vs untreated control cells: 100+/-10%; P<0.01). FACS analysis in NCI-H295 cells treated with either CLO or PAM demonstrated both apoptotic and necrotic cell death. However, while during PAM treatment cell number and hormone secretion decreased in parallel, we observed specific impairment of steroidogenesis in the presence of CLO with a dose-dependent increase in the 17-OH-progesterone/cortisol ratio (100 microM CLO 134+/-30%; 500 microM CLO 284+/-10%; 1000 microM CLO 545+/-130% vs 100+/-20% in control cells; P<0.01). Further analysis in ACTH-stimulated bovine adrenal cells using stable isotope dilution/gas chromatography-mass spectrometry demonstrated CLO-induced inhibition of adrenal 21-hydroxylase (P450c21) activity leading to a dose-dependent increase in the 17-OH-progesterone/11-deoxycortisol ratio. In conclusion, we demonstrate a dose-dependent antiproliferative effect of CLO and PAM in adrenocortical cells. In addition, for the first time, we describe a suppressive effect of CLO on steroidogenesis via inhibition of adrenal 21-hydroxylase (P450c21) activity.