Osteocalcin (OC), a noncollagenous bone matrix protein, is expressed in high levels by osteoblasts. To determine whether the OC promoter mediates cell-specific gene expression in cells of osteoblast lineage, we constructed a recombinant adenovirus, Ad-OC-TK, which contains the OC promoter that drives the expression of herpes simplex virus thymidine kinase (TK). We tested the expression of TK by this virus in osteoblast cell lines as well as in non-osteoblastic cell lines by assessing the enzyme activity of TK in vitro. Whereas the OC promoter failed to drive the expression of the TK gene in several non-osteoblastic cell lines such as WH, a human bladder transitional carcinoma, and NIH 3T3, an embryonic mouse fibroblast cell line, the OC promoter mediated high levels of expression in osteoblast cell lines including murine ROS and human MG-63 cells. The addition of acyclovir (ACV), a pro-drug for the inhibition of cell proliferation, resulted in the induction of osteoblast-specific cell death in vitro. Intratumoral injection of Ad-OC-TK into murine ROS osteosarcoma abolished tumor growth in a host treated with subsequent i.p. ACV injection in vivo. The Ad-OC-TK virus plus ACV treatment appears to be highly selective in blocking the growth of both murine and human osteosarcoma cell lines in vitro and murine osteosarcoma in vivo.