Numerous studies support the concept that the nonhemopoietic cells of the bone marrow (BM), are derived from a population of multipotent bone marrow stromal stem cells (BMSSCs), which reside in perivascular niches within the bone marrow. These BMSSCs are thought to give rise not only to more cells that are phenotypically and functionally identical but also differentiated, lineage-committed mesenchymal progeny, including chondrocytes, smooth muscle cells, adipocytes, and osteoblasts. Recently, we have generated a novel monoclonal antibody (mAb) (designated STRO-3) that reacts with a minor subset of STRO-1(+) cells contained within adult BM aspirates and does not react with CD34(+) hemopoietic stem cells. Our results also show that STRO-3 identifies a high proportion of BMSSCs that possess extensive proliferative and multilineage differentiative capacity. Using retroviral expression cloning, we determined that STRO-3 binds to tissue nonspecific alkaline phosphatase (TNSALP), a cell-surface glycoprotein usually associated with cells of the osteoblast lineage. Studies presented here suggest that in addition to being expressed by osteoblasts, TNSALP may also represent a marker of immature BMSSCs in vivo. Finally, these studies suggest that antibodies to TNSALP may be used as an effective single marker of enrichment of BMSSCs from various tissues.