Matrix metalloproteinases (MMPs) play a major role in the phenomena of growth, invasion, and metastasis of malignant disease. We studied the effects of doxycycline, a synthetic tetracycline that has been shown to suppress MMP activity in other solid tumors, on osteosarcoma (OSA) cell proliferation and MMP activity in vitro. OSA cells from 6 patients and from one established human tumor cell line (U2OS) (American Type Culture Collection) were cultured in the presence or absence of doxycycline. Doxycycline (10 microg/ml) suppressed OSA cell proliferation threefold to sevenfold in all cultures. MMP activity was assessed by gelatin zymography and was diminished by approximately 50% in all cultures. We examined the hypothesis that induction of apoptosis is one of the mechanisms by which doxycycline inhibits OSA cell proliferation. Ethidium bromide-stained gels of DNA from cells grown in the presence of 5 microg/ml and 10 microg/ml of doxycycline revealed laddering consistent with apoptosis after 24 hours in culture. The demonstration that doxycycline suppresses cell proliferation and MMP activity and induces apoptosis in human OSA cells in vitro suggests that this well-tolerated oral agent may be effective in the in vivo treatment of OSA.