Depletion and repletion of glutathione in two-cell to blastocyst stage mouse embryos was examined. Reduced (GSH) and oxidized glutathione (GSSG) were measured by fluorimetric HPLC after derivatization of extracted embryo samples with dansyl chloride. Addition of buthionine sulfoximine (BSO) to culture medium for 16 h decreased GSH levels in both two-cell and blastocyst stage embryos; however, GSH decreased more drastically in blastocysts. Addition of diethyl maleate (DEM) to culture medium depleted GSH in both two-cell and blastocyst stage embryos. After removal of DEM, GSH levels returned to normal in blastocysts, and addition of BSO or removal of cystine from medium blocked GSH repletion. Two-cell stage embryos were unable to recover GSH levels after depletion and exhibited decreased in vitro development. Addition of methionine to culture medium was unable to substitute for cystine as a source of cysteine in glutathione synthesis, indicating that the embryos do not use the cystathionine pathway. Embryos collected early on Day 3 of development were unable to recover GSH levels within 5 h, whereas embryos collected late on Day 3 recovered GSH within 5 h. Addition of cycloheximide to culture medium at noon on Day 3 of development decreased the ability of blastocysts to recover their GSH levels late on Day 3. These data indicate that GSH turnover and synthesis increases between the two-cell and blastocyst stages. The increase in the ability of embryos to synthesize GSH on Day 3 is dependent on protein synthesis. Cleavage stage embryos have limited capacity to synthesize GSH and appear susceptible to adverse effects of toxicants or conditions that deplete glutathione.