5-Fluorouracil (5-FU) inhibits the enzyme thymidylate synthetase (TS) which results in inhibition of DNA synthesis. 5-FU is teratogenic in many species, inducing cleft palate, limb, and tail defects. In the study, GD 14 embryonic rat palates were exposed to 5-FU in organ culture with increasing concentrations and durations of exposure. Palates exposed to 5-FU were morphologically abnormal and craniofacial shape, size, and palatal fusion pattern were affected with the severity of effects dependent on concentration and duration of exposure. Cleft palate was induced in vitro as opposing palates overlapped in a narrowed oral cavity. Palates exposed to higher levels of 5-FU were growth inhibited, but fused even though proliferation ceased and few cells were available to participate in elevation and fusion. This was demonstrated as a biphasic concentration-response profile for palatal fusion in which 0.05 to 0.15 micrograms 5-FU/ml produced decreasing rates of palatal fusion, while exposure to 0.15 to 3.0 micrograms/ml resulted in progressively increasing rates of fusion. The effects of 5-FU were detected biochemically as a reduction in TS activity which was concentration and time dependent during the first 12 hours. However, TS activity returned to control levels by 24 hours. During the first day, 5-FU did not alter protein levels, but DNA levels significantly decreased at 2.0 micrograms/ml. After 5 days in culture, DNA and protein both decreased with increasing 5-FU concentration and duration of exposure. There were no effects on cell cycle detectable by flow cytometric analysis, however 3H-TdR incorporation decreased in a concentration dependent manner. The study demonstrates that elevation and fusion can occur in the absence of growth and proliferation and that severe inhibition of growth or proliferation would not necessarily be sufficient to induce cleft palate.