To study the ultrastructure of regenerated bone, the ectopically formed bone induced by gelatin hydrogels incorporating bone morphogenetic protein 2 (BMP-2) was evaluated through transmission electron microscopic (TEM) analyses including bright field electron imaging, selected area diffraction (SAD) imaging, and dark field electron imaging. After 2 weeks of implantation, ectopic bone induction was apparent at Wistar rat thigh muscles receiving the hydrogels incorporating BMP-2 through soft X-ray observation. The specimens for TEM study were prepared with an anhydrous method using ethylene glycol to maintain the structure of hydroxyapatite crystals. TEM observation of unstained ectopic bone cross-sections showed that electron dense substances were deposited on the gap zone of collagen fibers (intrafibrillar crystallites), while needle-like electron dense substances were observed in the intermolecular space of collagen fiber (extrafibrillar crystallites). SAD patterns suggested that these electron dense substances were hydroxyapatite crystals. Furthermore, from the SAD pattern and dark field electron image of intrafibrillar crystallites, it was presumed that the crystallographic c-axis of apatite crystals oriented to the direction of collagen fibril.