A polyclonal antibody against rat osteocalcin propeptide (anti-pro-OC) has been prepared and used in immunohistochemical studies to demonstrate cells expressing osteocalcin (OC). With the anti-pro-OC antibody, predominant immunoreaction was observed in cuboidal osteoblasts along the bone formation surface, and in odontoblasts and cementoblasts of the tooth. The appearance of pro-OC antigenicity was well consistent with the expression of OC mRNA in the cells as demonstrated by in situ hybridization. Matrices around these cells showed no apparent pro-OC antigenicity, which contrasted remarkably with the immunoreaction with an antibody against the N-terminal sequence of mature rat OC (anti-OC-N). Although the anti-OC-N antibody positively reacted with OC-producing cells, the intensity of the immunoreaction was much weaker than that obtained with the anti-pro-OC antibody. Furthermore, we examined the appearance of pro-OC antigenicity in maxillary alveolar bone during experimental tooth movement in rats. When the maxillary first molar was moved to the mesial direction with a closed coil spring, the bone remodeling phase in the distal alveolar bone surface changed from dominantly resorptive to formative. After the application of orthodontic force, pro-OC-positive osteoblasts appeared on the distal alveolar bone surface (tension side), according to the appearance of alkaline phosphatase activity on the bone surface. Thus, the use of the anti-pro-OC antibody is a practical means to demonstrate cells actively expressing OC and could be an alternative to in situ hybridization.