The adhesive interactions that occur between bone cells and the developing matrix during bone formation help guide coupled remodeling and the maintenance of bone mass. Here, we provide evidence that acetylcholinesterase (AChE) is a novel osteoblast-derived mediator of cell-matrix interactions in bone. These findings complement an increasing body of evidence which suggests that AChE, in addition to its role in terminating cholinergic signaling, may be instrumental in regulating cellular differentiation and adhesion. We have shown, using RT-PCR, that osteosarcoma cell lines and primary cultures of osteoblasts express AChE mRNA. Expression appeared to be differentiation-dependent, and restricted to AChE splice variants containing the T subunit (exon 6). Immunofluorescent localization demonstrated that these osteoblastic cells expressed protein for AChE with an intracellular vesicular distribution. Immunohistochemistry on tissue sections confirmed AChE expression by osteoblasts in vivo, and revealed the presence of AChE along cement lines, also identified by enzyme histochemistry. In vitro functional studies indicated that osteoblast-like cells adhered specifically to and spread on AChE substrates, but did not interact with butyrylcholinesterase, a closely related protein. Our evidence strongly implicates AChE as a novel bone matrix protein, capable of mediating cell-matrix interactions, and as such may be a principal participant in organized bone formation and the regulation of remodeling.