Objectives: Our previous research demonstrated that vitamin A might be related to vocal fold development. The purpose of this study was to determine whether vitamin A deficiency affects prenatal laryngeal development in rats. Methods: Two considerations were necessary in designing a study using a rat model: For embryonic survival, vitamin A is necessary through day 10 of gestation, and laryngeal formation occurs primarily after day 11. Thus, we created a rat model that developed vitamin A deficiency after embryonic day 11. Ten pregnant rats (5 vitamin A-deficient rats and 5 control rats) were studied. Embryos were collected at embryonic day 18.5 and analyzed histologically. Results: Eighteen percent of the vitamin A-deficient embryos were alive and demonstrated laryngotracheal cartilage malformation, incomplete separation of the glottis, and/or laryngoesophageal clefts. Conclusions: These results document the important role played by vitamin A in laryngeal development.