Retinoic acid (RA) is teratogenic in many species and is an effective inducer of cleft palate in mice. The pathogenesis of cleft formation varies with the timing of exposure. It has been demonstrated, before formation of the palatal shelves, that RA exposure results in insufficient mesenchymal tissue, and palatal shelves fail to make contact. However, at the palatal shelf outgrowth stage, RA exposure affects shelf elevation and growth in rats, and possibly medial edge epithelium (MEE) differentiation in mice. The objective of this study was to examine the morphologic and functional changes associated with cleft formation in mice following exposure during shelf outgrowth. Particular emphasis was placed on evaluating the timing of palatal shelf elevation in RA exposed embryos and on identifying differentiation events occurring concurrently in the epithelium. On gestational day (GD) 12 (8:00 AM), gravid CD-1 mice were gavaged with 70 mg/kg RA or vehicle. This protocol produced a 100% incidence of cleft palate at term, allowing us to correlate the morphological and/or biochemical changes observed at pre-fusion time points. Embryos were collected at 12 hr intervals through GD 15, beginning 4 hr after exposure. Serial sections of embryos were either stained with H&E, with a battery of lectins [Sambucus nigra (SNA), Arachis hypogaea (PNA), Ricinus communis (RCA-1), Glycine max (SBA), Succinylated Wheat Germ (S-WGA)], or with a probe to hyaluronan. Throughout the period of normal palate development, the shelf mesenchyme showed increasing regional organization and progressive hydration and these changes were correlated with increase Hyaluronan (HA) deposition. RA treatment resulted in lose of regional organization and delayed mesenchyme hydration. In association with these changes there were reductions in HA deposition and extracellular matrix glycoconjugates recognized by PNA in the palate mesenchyme. Further there was a considerable delay in palatal shelf elevation and palate shelf did not make contact at the midline. Our data indicates, in embryos exposed on GD 12 to levels of RA sufficient to induce a 100% incidence of clefting, that cleft formation is a result of palatal shelves failing to make contact. Alterations in mesenchyme development and the subsequent delay in palate shelve elevation are central to RA-induced cleft formation following exposure at the palate shelf out growth stage.