Alkaline phosphatase (AP), which is located in the matrix vesicles originated in osteogenic cells, plays a key role in calcifying bone and cartilage. In this work we standardized a method of extracting a membrane fraction rich in alkaline phosphatase (MFAP) from cultures of human alveolar bone cells. Human alveolar bone fragments (explants) discarded from surgical procedures were transferred into a sterile centrifuge tube and digested during 6 cycles of 30 min, with 1 mg/ml of collagenase type II at 37ºC in a water bath under constant agitation. The supernatant fractions numbers one and two were discarded and fractions numbers three to six were transferred to another centrifuge tube containing an equal amount of culture medium and centrifuged at 200 g for 5 minutes. The bone-derived cells and remaining explants were combined and cultured in α-MEM, supplemented with 10% fetal bovine serum, 50 µg/ml gentamicin, 0.3 µg/ml fungizone, 5 µg/ml ascorbic acid, 7 mM β-glycerophosphate, and 10^-7 M dexamethasone in 75 cm² culture flasks, until confluence at 37ºC and 5% CO₂. After 21 days of growth, the cultures presented osteoblast-like cells and mineralizing matrix, which could be observed when the culture was stained with Alizarin red S.