We have determined effect of the oxidant peroxynitrite (ONOO-) on Ca2+-dependent matrix metalloprotease-2 (MMP-2) activity and the role of the protease on Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane under ONOO- -triggered conditions. The smooth muscle plasma membrane possesses a 72-kDa protease activity in a gelatin-containing zymogram. The 72-kDa protease activity has been found to be inhibited by tissue inhibitor of metalloprotease-2 (TIMP-2), indicating that the protease is the matrix metalloprotease-2 (MMP-2). Treatment of the membrane suspension with ONOO- caused stimulation of the MMP-2 activity (as evidenced by 14C-gelatin degradation) and also increased Ca2+ ATPase activity. The ONOO- -triggered protease activity and the Ca2+ ATPase activity were found to be inhibited by the antioxidants: vitamin E, thiourea, and mannitol. Pretreatment with catalase and superoxide dismutase did not significantly alter ONOO- -stimulated MMP-2 activity and Ca2+ATPase activity, indicating that peroxide and superoxide are not present in appreciable amount in ONOO-. Under both basal and ONOO- triggered conditions, the MMP-2 activity and the Ca2+ ATPase activity were also inhibited by EGTA, 1:10-phenanthroline, and TIMP-2. However, the ONOO- -stimulated MMP-2 activity and the Ca2+ ATPase activity were found to be insensitive to phenylmethylsulfonylfluoride, Bowman-Birk inhibitor, chymostatin, leupeptin, antipain, N-ethylmaleimide, and pepstatin. These results suggest that ONOO- caused stimulation of MMP-2 activity and that the increased MMP-2 activity subsequently played a pivotal role in stimulating Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane.