Retinoic acid (RA) is essential for normal embryonic development, but excess RA during pregnancy can cause various congenital anomalies such as craniofacial and limb malformations both in humans and rodents. We reported in a previous study that exogenous RA might prevent tongue withdrawal and elevation of palatal shelves in mouse fetuses, resulting in cleft palate. In the present study, we observed the morphogenesis of the tongue and the differentiation of tongue intrinsic muscles in mouse fetuses exposed to exogenous RA in utero. The upper median surface of the tongue was depressed and notches were observed in the bilateral edges in RA-treated fetuses. Intrinsic muscle fibers of the tongue were also bent at the midline. Such aberrant morphogenesis of the tongue in RA-treated fetuses was never observed in control fetuses. Both RA-degrading enzymes (Cyp26a1 and Cyp26b1) and one of the RA-synthesizing enzymes (retinaldehyde dehydrogenase2: RALDH2) were expressed at early stages of normal tongue development, and RA caused remarkable up-regulation of Cyp26a1 and Cyp26b1. The expression of Cyp26a1 was hardly observed in the tongue primordia on early E11 by in situ hybridization on sections. However, in RA-treated fetuses, the robust expression of Cyp26a1 was observed in the epithelium of the tongue primordia as early as E11 and even in the tongue epithelium and mesenchyme ectopically at late E11. Cyp26b1, another isozyme of Cyp26s, began to be expressed early on E11 in the tongue of control fetuses, although its expression was restricted to the mesenchyme near the median sulcus of the tongue primordium. However, the expression of Cyp26b1 increased significantly in the mesenchyme, especially in that of the caudal part of the tongue in RA-treated fetuses at early E11 and its up-regulation continued until late E12. That is, Cyp26a1 is ectopically up-regulated in response to excess RA, while Cyp26b1 is up-regulated in response to excess RA in the region where it is expressed during normal development. RALDH2 expression in the fetal tongue was not affected by exogenous RA. We examined the RA distribution in the fetal tongue by b-galactosidase staining controlled by a sensitive RA response element (RARE), using RARE-hsplacZ reporter mice. RARE-hsplacZ positive cells was observed both in tongue intrinsic and extrinsic muscles in normal fetuses, but in RA-treated fetuses, it was not observed in tongue muscles but exhibited a diffuse distribution in the tongue mesenchyme. We also found that Tbx1, a candidate gene of DiGeorge/velocardiofacial syndrome (DGS/VCFS), was down-regulated in the fetal tongue in response to excess RA. Tbx1 was expressed in the genioglossus muscle primordium from E12.5 and down-regulated by excess RA. In the previous study Tbx1 was shown to regulate myogenesis in craniofacial development and clinically DGS/VCFS patients often have problems in feeding and swallowing, which may be due to poor coordination of the tongue, pharyngeal, and esophageal muscles. On the other hand, RA has been known to promote skeletal muscle development by stimulating myogenesis in myotomes in zebrafish. Therefore, our study strongly suggests that RA signaling plays an important role in tongue muscle development via regulation of Tbx1 and that its perturbation may induce abnormal tongue development. Because impaired tongue muscle development can result in various problems in feeding, swallowing and speech, further studies are surely needed to elucidate the molecular mechanism of normal and abnormal tongue development.