Msx2 is a homeodomain transcription factor that regulates craniofacial development in vivo and osteocalcin (Osc) promoter activity in vitro. Msx2 is expressed in many craniofacial structures prior to embryonic day (E) E14 but is expressed at later stages in a restricted pattern, primarily in developing teeth and the calvarium. We examine Osc expression by in situ hybridization during murine development, detailing temporospatial relationships with Msx2 expression during preappositional and appositional odontogenesis and calvarial osteogenesis. Osc expression at E14-14.5 is very low, limited to a few perichondrial osteoblasts in the dorsal aspect of developing ribs. At E16.5 and E18.5, Osc expression is much higher, widely expressed in skeletal osteoblasts, including calvarial osteoblasts that do not express Msx2. No Osc is detected in early preappositional teeth that express Msx2. In incisors studied at an early appositional phase, Msx2 is widely expressed in the tooth, primarily in ovoid preodontoblasts and subjacent dental papilla cells. Osc is detected only in a small number of maturing odontoblasts that also express alpha1(I) collagen (Colla1) and that are postproliferative (do not express histone H4). Msx2 expression greatly overlaps both histone H4 and Colla1 expression in ovoid preodontoblasts and dental papilla cells. By the late appositional phases of E18.5 and neonatal teeth, Osc mRNA is highly expressed in mature columnar odontoblasts adjacent to accumulating dentin. In appositional bell-stage molars, reciprocal patterns of Msx2 and Osc are observed in adjacent preodontoblasts and odontoblasts within the same tooth. Osc is expressed in mature columnar odontoblasts, while Msx2 is expressed in adjacent immature ovoid preodontoblasts. In less mature teeth populated only by immature ovoid preodontoblasts, only Msx2 is expressed-no Osc is detected. Thus, Msx2 and Osc are expressed in reciprocal patterns during craniofacial development in vivo, and Msx2 expression in preodontoblasts clearly precedes Osc expression in odontoblasts. In functional studies using MC3T3-E1 calvarial osteoblasts, Msx2 suppresses endogenous Osc, but not osteopontin, mRNA accumulation. In toto, these data suggest that Msr2 suppresses Osc expression in the craniofacial skeleton at stages immediately preceding odontoblast and osteoblast terminal differentiation.