Members of the TGF-beta superfamily are secreted signaling proteins that regulate many aspects of development including growth and differentiation in skeletal tissue. There are three isoforms of TGF-beta that act through the same heteromeric receptor complex. To address the question of the role of TGF-beta signaling in skeletal development, we generated mice with a conditional deletion of the TGF-beta type II receptor gene (Tgfbr2) specifically in Col2a expressing cells using the Cre/lox recombinase system. Alizarin red-/Alcian blue-stained skeletons were prepared from embryos at 17.5, 15.5, and 13.5 days of gestation. Col2acre+/-;TgfbrloxP/loxP and Col2acre-/-;Tgfbr2+/loxP skeletons were compared. Multiple defects were observed in the base of the skull and in the vertebrae. Specifically, the size and spacing of the vertebrae were altered, and defects were detected in the closure of the neural arches. In addition, alterations in transverse processes, costal joints, and zygapophyses were detected. While the vertebral bodies were only moderately affected, the intervertebral discs (IVDs) were either missing or incomplete. Alterations in the vertebrae could be detected as early as E13.5 days. Surprisingly, alterations in length and mineralization of long bones were not detected at E17.5 days. In addition, the expression patterns of markers for chondrocyte differentiation were not altered in vertebrae or long bones suggesting that loss of responsiveness to TGF-beta in chondrocytes does not affect embryonic endochondral bone formation. In contrast, mice that survived postnatally demonstrated alterations in the length of specific bones. Skeletons from Col2acre+/-;Tgfbr2loxP/loxP mice were compared to those from mice null for the TGF-beta2 ligand. The differences observed between these models allow distinctions to be made between the roles of the various isoforms of TGF-beta and the signaling in specific cell types. The data provide information regarding mechanisms of skeletal development and suggest that TGF-beta signaling is a critical component.