The recent report of a transforming growth factor-beta 3 (TGF-beta 3) knock-out mouse in which 100 percent of the homozygous pups have cleft palate raised the question as to the potential usefulness of these animals as a model for cleft palate research. The specific aim in this study was to carefully document the anatomy of the cleft palate in the TGF-beta 3 knock-out mice as compared with wild type controls. Special attention was paid to the levator veli palatini muscle, the tensor veli palatini muscle, and their respective innervation. Because the TGF-beta 3 knock-out is lethal in the early perinatal period and because the heterozygotes are phenotypically normal, polymerase chain reaction was required to genotype the animals before mating. Time-mated pregnancies between proven heterozygotes were then delivered by cesarean section at gestational day 18.5 to prevent maternal cannibalism of homozygote pups. All delivered pups were killed and their tails processed by polymerase chain reaction to verify genotype. The heads were then fixed and sectioned in axial, coronal, or sagittal planes. Sections were stained with hematoxylin and eosin or processed for immunohistochemistry with nerve specific protein gene product 9.5 and calcitonin gene-related peptide antibodies. Sections were analyzed in a serial fashion. Nine wild type control animals were analyzed along with nine TGF-beta 3 knock-out homozygotes. Time matings between proven heterozygotes yielded wild type pups, heterozygote pups, and homozygote knock-out pups in the expected mendelian ratios (28 percent to 46 percent to 26 percent; n = 43). The results demonstrated 100 percent clefting in the homozygous TGF-beta 3 knock-out pups. Complete clefting of the secondary palate was seen in four of nine and incomplete clefting was seen in five of nine. The levator veli palatini and tensor veli palatini muscles were demonstrated coursing parallel to the cleft margin in all cleft mice. The orientation of these muscles differs from the normal transverse sling of the levator veli palatini muscle and the normal palatine aponeurosis of the tensor veli palatini muscle at the soft palate in control animals. Innervation of the levator veli palatini muscle by cranial nerve IX and the tensor veli palatini muscle by cranial nerve V were demonstrated in both cleft and control animals by use of immunohistochemistry with nerve-specific antibodies. Demonstration of a teratogen-free, reproducible animal model of clefting of the palate with a known, single-gene etiology is an important step in the systematic understanding of a congenital defect whose multifactorial etiology has hampered previous research efforts. This study presents a detailed anatomic description of such a model, including a description of the muscular anatomy and the innervation of the muscles of the palate. Because of early perinatal mortality, this model has limited applications for postnatal studies.