A procedure for the in vitro cultivation of embryonic rat palatal tissue, using semi-defined media, is described. Palatal fusion obtained in vitro resembles that normally seen in vivo. The effect of 6-aminonicotinamide (6 AN) a niacin inhibitor and of excess vitamin A on palatal fusion was investigated using this in vitro system. Administration of 6 AN to the mother resulted in a high incidence of failure to fuse and of partial fusion. This compound appeared to produce clefts by interfering with the ability of the shelves to fuse but not otherwise affecting their development. This effect was prevented by the simultaneous administration of nicotinamide to the mother. Addition of 6 AN to the culture medium was less effective and was not reversed by addition of nicotinamide to the medium. Histological changes were seen in most treated palates regardless of the route of administration of 6 AN. These changes could be prevented by nicotinamide, either injected or added to the culture medium, but no relation could be found between the tissue changes and the occurrence of clefts. Addition of vitamin A to the culture medium resulted in a high incidence of failures of palates to fuse. This appeared to be due to retarded development of the shelves, with failure to meet in the midline. Experiments with sliced palatal preparations indicated that the tissues were competent to fuse if they were able to come together. Alcian Blue staining and autoradiography with 35SO4 did not indicate that mucopolysaccharide synthesis or distribution or sulfur metabolism are factors involved in the vitamin A effect. Evidence is presented supporting the view that palatal fusion may be interrupted by nutritional factors operating through different mechanisms, although the end result observed at birth (cleft palate) is the same.