The effects of β-mercaptoethanol (β-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O₂ supplemented with β-ME. Addition of β-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O₂, to almost the same rates when they were cultured in 5% O₂. To investigate whether the growth-promoting effect of β-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without β-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without β-ME. In contrast, addition of β-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by β-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by β-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of β-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that β-ME has a protective effect in embryo development against oxidative stress and that the effect of β-ME is associated with the promotion of cystine uptake of low availability in embryos.