The effect of p-nitrophenyl beta-D-xyloside, an inhibitor of proteoglycan biosynthesis, on the growth of chick embryos was studied by injection of the single dose of 1.0 mg/egg into fertile eggs on day 3. Embryos examined on day 10 had systemic edema, and were increased not only in wet weight (142% of the non-treated embryos) but also in dry weight (125%). No skeletal malformations were observed in the treated embryos. The glycosaminoglycan content in the treated embryos began to increase 6 h after treatment and reached the maximum level (174% of the non-treated) after 3 days, while the DNA and protein content began to increase 12 h after treatment and reached the maximum level (about 140%) within 3 days, p-Nitrophenyl alpha-D-xyloside, p-nitrophenyl beta-D-galactoside, and a mixture of p-nitrophenol and D-xylose produced neither the abnormal overgrowth nor the edematous change of chick embryos. When fertile eggs were treated with 1.0 mg/egg of p-nitrophenyl beta-D-xyloside on day 6, the increase in wet and dry weights was also observed in all surviving embryos. On the contrary, treatment on day 9 resulted in the slight reduction of embryonic growth in addition to the systemic edema. Both embryos treated on day 3 and on day 6 contained glycosaminoglycans rich in chondroitin 6-sulfate, whereas the embryos treated on day 9 contained glycosaminoglycans rich in undersulfated chondroitin sulfates. These findings seem to support the view that glycosaminoglycans play an important role in the regulation of embryonic growth.