OBJECTIVE: Recent studies have demonstrated a positive effect of a low oxygen concentration on reproductive performance of human cleavage-stage embryos and blastocysts. Since many IVF centers perform embryo transfers on day 3, our goal was to address the effect of low oxygen concentration at this developmental stage, and its subsequent pregnancy outcome. DESIGN: Prospective, randomized. MATERIALS AND METHODS: We conducted a prospective randomized simple blind study to compare in vitro embryo quality and pregnancy rate using two different oxygen concentrations in air. One hundred and eighty one IVF/ICSI cycles were randomly assigned to a two-gas incubator system (6% CO2 and 19% O2 in air) or three-gas incubator system (6% CO2, 5% O2 and 89% N2). Groups were comparable regarding female age, ovarian reserve, infertility cause and ovarian hyperstimulation protocols. There were also no differences in number of metaphase II oocytes or number of embryos transferred between groups. Embryos were scored just prior to transfer on day 3 using conventional morphology classification. Statistical tests were performed using Fisher's exact or t tests as appropriate. RESULTS: A total of 181 IVF/ICSI cycles were randomized to both incubators, resulting in 144 embryo transfers (72 on each arm). Three hundred and twenty one embryos were cultured in the two-gas system and 325 in the three-gas system. Fertilization and cleavage rates were similar in both groups, as well as embryo quality distribution and implantation rates. Ongoing pregnancy rates tended to be higher in the three-gas system than in the two-gas system (30.5% vs. 22.2% respectively), although the difference was not significant (p=0.34). CONCLUSION: We found no differences in day 3 embryo quality using low oxygen culture. Pregnancy rates, although higher in the three-gas system, needs further confirmation with increased data sets.