Recent studies have demonstrated the protective effects of supplementing free oxygen radical scavenging enzymes against hyperglycemia-induced embryonic malformations. In this study, the glutathione (GSH)-dependent protection system in hyperglycemia-induced embryopathy was investigated. Rat embryos at the early head-fold stage (day 9.5) cultured in 66.7 mmol/l glucose for 48 h showed significant growth retardation and an increase in the frequency of malformations. The concentration of GSH and activity of the rate-limiting GSH-synthesizing enzyme, γ-glutamylcysteine synthetase (γ-GCS), significantly decreased in embryos exposed to hyperglycemia compared with controls (7.9 ± 0.6 vs. 12.5 ± 0.9 nmol/mg protein, P < 0.01 and 13.3 ± 1.9 vs. 22.6 ± 1.1 μU/mg protein, P < 0.01, respectively). Decreased activity of γ-GCS in embryos exposed to hyperglycemia was associated with decreased expression of γ-GCS mRNA levels. However, the activities of superoxide dismutase and glutathione peroxidase did not significantly change in these embryos. Extracellular and intracellular free oxygen radical formations estimated by Lucigenin-dependent chemoluminescence and flow cytometric analysis using 2′,7′-dichlorofluorescein diacetate increased in isolated embryonic cells taken from embryos cultured under hyperglycemia. Supplementation of 2 mmol/l GSH ester into the hyperglycemic culture nearly restored GSH concentration in these embryos (11.9 ± 0.5 vs. 12.5 ± 0.9 nmol/mg protein) and reduced the formation of free oxygen radical species leading to almost complete normalization of growth retardation and embryonic dysmorphogenesis. These results indicate that the GSHdependent protection system has a central role against oxidative stress in embryos cultured under hyperglycemia and that GSH depletion in embryonic cells during the critical periods of organogenesis plays a role in hyperglycemia-induced embryopathy.