Maternal biotin deficiency is strongly teratogenic in CD-1 mice. The most common malformations are craniofacial and limb defects such as cleft palate, micrognathia and micromelia. The effect of biotin deficiency on palatal development in mouse embryos on d 12 of gestation was studied by culturing mouse embryonic palates in serum-free medium using a suspension culture system. In control embryos palatal processes developed to the fused stage after 72 h in culture. The fusion of palatal processes was further increased by the addition of biotin (10(-8) mol/L) to the medium. The addition of organic acids such as propionic, beta-methyl crotonic or beta-hydroxy isovaleric acids as well as avidin to the medium did not affect the stage of palatal formation. Cycloheximide completely blocked the fusion of palatal shelves. In embryos from biotin-deficient mice, the incidence of fusion between the palatal shelves was < 7% and increased to > 30% when biotin (10(-8)-10(-6) mol/L) was added to the medium. The addition of fatty acids to the organ culture medium did not have any effect on the fusion of palatal processes. The incorporation of 35S-methionine into protein from biotin-deficient embryo explants was 88% of that in controls. The results indicate that biotin deficiency may interfere directly with synthesis of specific proteins and the formation of palatal processes.