Fetal rat limbs were obtained from pregnant females on the 14th day of gestation. Forelimbs and hindlimbs were pooled separately and preincubated for 22 hours at 37°C in Eagle's MEM containing the teratogen and folic acid antagonist, 9-methyl pteroylglutamic acid (9-mePGA). Following removal of old medium, fresh medium together with the teratogen were added at the same concentration as before. Tritiated glucosamine (10 μCi) was added to each vial and the incubation continued for 4 hours at 37°C. Tissues were homogenized, digested with pronase, and glycosaminoglycans (GAG) selectively precipitated with cetylpyridinium chloride. In vitro exposure of fetal limbs to various doses of 9-mePGA resulted in a significant increase in the accumulation of labeled GAG by fetal forelimbs at the high and intermediate dosage levels. No effect was observed at the lowest dose. Accumulation of labeled GAG by fetal hindlimbs although similar to fetal forelimbs in control incubations, was unaffected by in vitro exposure to 9-mePGA at any of the dosage levels studied.