Accumulating evidence indicates that calcification by isolated mammalian matrix vesicles (MVs) can be initiated by ATP. Since ATP can be hydrolyzed by either a specific ATPase or by nonspecific alkaline phosphatase (ALP), it remains to be established whether ATPase or ALP mediates ATP-initiated Ca and Pi deposition. To support the hypothesis that specific ATPase is responsible for ATP-initiated calcification by MVs isolated from mammalian cartilage and bone, the effects of ATP analogs, ALP substrates, and specific inhibitors on ATP hydrolysis and ATP-initiated calcification were compared between intact MVs and monoclonal antibody affinity-purified MV ALP. ATP analogs such as ADP and AMP exerted marked inhibitory effects on both [gamma-32P]ATP hydrolysis and ATP-initiated calcification by intact MVs, whereas phosphomonoesters such as beta-glycerophosphate or phosphoethanolamine had no effect. In contrast to intact MVs, purified MV ALP failed to calcify, and its [gamma-32P]ATP hydrolytic activity was readily inhibited by phosphomonoesters. Additionally, [gamma-32P]ATP hydrolysis by purified ALP in contrast to that by intact vesicles was completely inhibited by l-tetramisole, a specific inhibitor of ALP, suggesting a loss of specific ATPase during purification. Vanadate inhibition of ATP hydrolysis by purified ALP can be decreased by increasing ATP concentrations. On the contrary, ATP concentrations did not affect vanadate inhibition of ATP hydrolysis by intact MVs if ALP activity was blocked by l-tetramisole. These observations, therefore, suggest that: 1) a portion of [gamma-32P]ATP hydrolysis by MVs is attributable to a specific ATPase, whereas the remaining activity is due to ALP; and 2) a specific ATPase, but not ALP, is responsible for ATP-dependent Ca- and Pi-depositing activity of MVs isolated from bone or cartilage.