Bone formation in vivo occurs via two major processes, one of which depends on pre-existing cartilage, and the other does not. Bone morphogenetic proteins (BMPs) have been suggested to induce cartilage formation from non-skeletogenic mesenchymal cell population, which results in osteogenesis through the endochondral sequence. In the present study we examined if BMPs could cause direct bone formation independent of pre-existing cartilage using bovine fibrous collagen membrane (FCM) as a carrier for BMPs. Bovine metatarsal bone was extracted in 4 M guanidine HCl and BMPs were partially purified through the hydroxyapatite chromatography and the Heparin-Sepharose CL6B chromatography. The carrier was loaded with BMPs and then implanted in Wistar rats subcutaneously. The implants were fixed together with surrounding tissue every week after implantation and processed for von Kossa stain, immunohistochemistry, and electron microscopy. The phenotypes of bone and cartilage were identified histologically and immunohistochemically using antibodies against type I and type II collagen. Cartilage and bone were independently induced by 2 weeks. The bone formed directly on the collagen substrate of FCM without pre-existing cartilage. Calcification occurred in the carrier as well as the cartilage and bone matrix. The present study suggests that the BMPs induce osteogenesis in vivo independent of the endochondral sequence. © 1993 Wiley-Liss, Inc.