Matrix vesicles were isolated from epiphyseal growth plates of young rabbits. Lactate dehydrogenase activity was detected in the isolated matrix vesicles only in the presence of detergents, suggesting that NADH, the cofactor for the assay, does not penetrate the membrane of matrix vesicles. In contrast, the activity of alkaline phosphatase, a marker enzyme of the outer surface of matrix vesicles, was detected in the matrix vesicles using p-nitrophenyl phosphate as the substrate both in the presence and absence of detergents. Lactate dehydrogenase activity was detected only in the cytosol of chondrocytes of the epiphyseal growth plates but not in other subcellular fractions, showing that lactate dehydrogenase is not from the plasma membrane and membranes of intracellular organelles of chondrocytes. The isolated matrix vesicles contained all five lactate dehydrogenase isoenzymes but did not possess other cytosolic enzymes. These results show that lactate dehydrogenase is located in the matrix vesicles and suggest the presence of a mechanism for the specific uptake of cytosolic lactate dehydrogenase and the possibility of enzymatic quantification of the matrix vesicles at various calcification sites.