Bone regeneration occurs as a series of events that requires temporal and spatial orchestration of numerous cell types guided by the transcriptional activity of thousands of genes, as recently demonstrated by our laboratory. Using the rat femoral fracture model, bioinformatics, cloning, expression assays, fusion proteins, and transfection, we report on the identification and characterization of one such differentially expressed gene, termed Mustang (musculoskeletal temporally activated novel gene). Mustang encodes for an 82 amino acid nuclear protein with no homology to any known protein family. However, other species homologues (mouse, human, cow) were identified within EST (expressed sequence tag) databases. Nuclear localization was confirmed using a GFP-Mustang fusion protein. Using in situ hybridization, Mustang expression was localized to differentiating periosteal osteogenic cells, proliferating chondrocytes, and osteoblasts of the fracture callus. Unlike adult tissues, developing embryos abundantly express Mustang, especially in mesenchymal condensations of limbs, vertebral perichondrium, and mesenchymal cells of the intervertebral discs. Although the precise function of Mustang is unknown, its unique pattern of expression during bone development and regeneration, absence in adult tissues (except skeletal muscle and tendon), and nuclear localization suggest that Mustang is involved in the development and regeneration of the mammalian musculoskeletal system.