Fetal bovine mandible-derived osteoblasts were cultured for the purpose of obtaining a spatiotemporal assessment of bone matrix protein expression during in vitro differentiation. The results obtained from electron microscopic, immunohistological, biochemical, and molecular biological analyses indicated that these primary cultured osteoblasts produce an abundant extracellular matrix which mineralizes during a 14-day culture period. During this process, a restricted, spatiotemporal pattern of bone sialoprotein expression was indicated by immunohistological and molecular evaluations. To test the possibility that bone sialoprotein promoted the continued morphodifferentiation of osteoblastic cells, cultures were grown in the presence of anti-bone sialoprotein antibodies known to interfere with cell-bone sialoprotein attachment. Compared with cultures grown in the presence of normal rabbit serum (1:150), cultures grown in the media containing anti-bone sialoprotein antibody (1:150) failed to mineralize as demonstrated by von Kossa staining and failed to express osteocalcin and osteopontin as shown by the reverse transcription polymerase chain reaction. These results contribute to the growing evidence that bone sialoprotein is an important determinant of osteoblast differentiation and bone formation. Matrix protein-cell interactions may be examined using this spatiotemporally defined model.