Equine embryos appear to require a high glucose concentration for development to the blastocyst stage. The complete cell-culture medium, DMEM/F-12 (DM), which contains 17 mm glucose, has been widely used for equine embryo culture; however, in other species, high glucose during the early stages of embryo development is detrimental. To avoid this, we initiated a 2-step system using a low-glucose human embryo culture medium (Global) from Days 0 to 5 [Day 0 = day of intracytoplasmic sperm injection (ICSI)], with glucose (20 mm) added to the medium in the second step (Days 5 to 10; Choi et al. 2015 Reproduction 150, 31–41). We noted a high pregnancy loss rate (20%) in our clinical ICSI program (Hinrichs et al. 2014 J. Equine Vet. Sci. 34, 176), which used this 2-step Global system. Limited data are available on pregnancy with DM-produced embryos, but in one study, the loss rate was 1/13 (7.7%; Choi et al. 2011 Reproduction 142, 529–538). It is possible that use of DM in the second step of culture would better support normal blastocyst development than does Global with added glucose. However, DM is typically used at 5% CO₂, and Global at 6% CO₂, so use of both media would necessitate 2 sets of incubators. In the present study, we explored the use of DM in the second step of a two-step equine embryo culture system, under different CO₂ environments. Oocytes were collected from research mares via follicle aspiration and were held overnight before being matured in vitro for 30 h. All media included 10% fetal bovine serum. On Days 0 to 5 after ICSI, all embryos were cultured in Global under 6% CO₂ in mixed gas (5% O₂ and remainder N₂) at 38.2°C. In Experiment 1, on Day 5, embryos were transferred to DM prepared according to our standard method, with 14.3 mm NaHCO₃ and 5 mm NaOH, and were cultured in mixed gas at either 5% CO₂ or 6% CO₂. Five replicates were performed. In Experiment 2, DM was prepared by our standard method, or with 24.2 mm bicarbonate and no NaOH. When pH was measured using a pH meter after media were equilibrated overnight, this higher bicarbonate provided the same pH at 6% CO₂ (pH 7.3), as was achieved with the standard DM preparation at 5% CO₂. Six replicates were performed. In both experiments, blastocyst development was assessed on Days 7 to 10, and blastocyst rates were compared between treatments by Fisher’s exact test. In Experiment 1, blastocyst rates were 43%, 13/30 and 27%, 8/30 for the standard DM preparation in 5% and 6% CO₂, respectively (P > 0.05). In Experiment 2, the blastocyst rates were 34%, 14/44 for the standard DM preparation at 5% CO₂ and 43%, 19/44 for the high-bicarbonate DM at 6% CO₂ (P > 0.05). We conclude that a 2-step Global-DM system can support equine blastocyst production under a consistent CO₂ environment (6%) if DM bicarbonate levels are adjusted to balance the increased CO₂.