AbstractAll known bone‐derived osteoinductive factors have been isolated from endochondral (EC) bones and all initiate bone induction via EC ossification. However, to date no attempt has been made to isolate comparable factors from bones which form initially and completely via intramembranous (IM) ossification. The purpose of this work was to isolate osteoinductive proteins from IM bones. To accomplish this, we extracted proteins from bovine frontal bone matrix (intramembranous origin) using methods previously described for endochondral (EC) bone matrix (i.e., femur). Bone powder (<1 mm) was decalcified and proteins extracted with 4 M guanidine hydrochloride. Ultrafiltration was used to isolate and concentrate a 10–100 kilodalton (kDa) fraction, upon which heparin‐Sepharose (HS) affinity chromatography was performed. HS‐binding (HS‐B) and non‐binding proteins (HS‐NB) were lyophilized with bovine type I collagen (Vitrogen) to form pellets which were implanted subcutaneously in rats. Radiology as well as brightfield, fluorescent, and polarizing microscopy were used to assess the formation of ectopic bone at the site of pellet implantation. In this report we demonstrate that a heparin‐Sepharose binding, osteoinductive factor can be extracted and partially purified from bovine intramembranous bone matrix. This factor has a different sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) banding pattern than a comparable osteoinductive/chondroinductive factor isolated from EC bone. © 19