A monoclonal antibody raised against alkaline phosphatase (ALP) of human osteosarcoma was used to localize this enzyme in human fetal bone tissue. For light microscopy, the presence of alkaline phosphatase in osteoblasts and osteocytes was demonstrated by use of an avidin-biotin immunoperoxidase procedure. Electron microscopic immunolocalization was accomplished with an indirect immunoperoxidase method which revealed a concentration of the enzyme on matrix vesicle and osteoblast plasma membranes. In addition, many vesicular protrusions arising from areas of plasma membrane on the lateral surfaces of adjacent osteoblasts were strongly immunolabeled. Immunostaining for ALP was absent in vesicles which contained fine crystallites. Alkaline-phosphatase-rich matrix vesicles may play a significant role in the mineralization of the extracellular matrix.