Recently, it has become increasingly evident that fracture healing involves a complex interaction of many local and systemic regulatory factors. The roles of some of these growth factors have been described; however, little is understood about the presence of the bone morphogenetic proteins in fracture repair, despite the fact that they are the most potent osteoinductive proteins known. This study defines and characterizes the physiologic presence, localization, and chronology of the bone morphogenetic proteins in fracture healing with an established rat fracture healing model. With use of a recently developed monoclonal antibody against bone morphogenetic proteins 2 and 4 developed with standard avidin-biotin complex/immunoperoxidase protocols, frozen undecalcified fracture calluses were analyzed semiquantitatively for the percentage of various types of fracture cells staining positively. During the early stages of fracture healing, only a minimum number of primitive cells stained positively in the fracture callus. As the process of endochondral ossification proceeded, the presence of bone morphogenetic proteins 2 and 4 increased dramatically, especially in the primitive mesenchymal and chondrocytic cells. While the cartilaginous component of the callus matured with a concomitant decrease in the number of primitive cells, there was a concomitant decrease in both the intensity and the number of positively staining cells. As osteoblasts started to lay down woven bone on the chondroid matrix, these osteoblastic cells exhibited strong positive staining. The intensity of this staining decreased, however, as lamellar bone replaced the primitive woven bone. A similar observation was noted for the areas of the callus undergoing intramembranous ossification. Initially, within several days after the fracture, periosteal cells and osteoblasts exhibited intense staining for bone morphogenetic proteins 2 and 4. As the woven bone was replaced with mature lamellar bone, this staining decreased. These data, and the awareness of the strong osteoinductive capacities of bone morphogenetic protein, suggest that bone morphogenetic proteins 2 and 4 are important regulators of cell differentiation during fracture repair.