We have shown that yeast pyrophosphatase dissolves calcium pyrophosphate dihydrate (CPPD) crystals in solutions. In this investigation we demonstrate that alkaline phosphatase (ALP) effectively dissolves CPPD crystals in vitro. CPPD dissolution by ALP had a pH optimum of 7.4, which is the optimum pH for its pyrophosphatase (PPiase) activity. The CPPD dissolution and PPiase activity by ALP are magnesium dependent, whereas its phosphoester hydrolytic activity is not. Calcium, which inhibited the enzymatic CPPD dissolution and PPiase activity of ALP had no effect on its phosphoester hydrolytic activity. These data indicate that PPiase activity of ALP is responsible for CPPD dissolution and not its phosphoester hydrolytic activity. Matrix molecules such as proteoglycans and chondroitin sulfate had no effect on the enzymatic and nonenzymatic dissolution of CPPD crystals. ALP acted more effectively on CPPD crystals than on soluble pyrophosphate relative to yeast PPiase. Our data suggest that chondrocyte ALP may play an important role in the dissolution of CPPD crystals in cartilage.