Embryos were collected from superovulated Dutch rabbits 19 h after injection of LH and insemination. The embryos were at the one-cell stage at that time and those judged to be normal by the absence of granular cytoplasm and regular shape were distributed randomly within donors into culture dishes containing 500 μl of a macromolecule-free medium consisting of RPMI-1640 and low glucose Dulbecco's modified Eagle's medium, 1:1, without a cover of oil. In Expt 1, O ₂ concentrations of 5, 10 and 15%, with 5% CO ₂ plus 90, 85 and 80% N ₂ , respectively, were tested. In Expt 2, O ₂ concentrations of 1, 5 and 20% were combined with 5% CO ₂ and the remaining gas was N ₂ . After 84 h in culture at 39°C, embryos were examined for stage of development and stained with Hoechst 33342 so that the number of cells could be counted. In Expt 1, the proportion of embryos reaching the hatching blastocyst stage after 84 h in culture in 5, 10 and 15% O ₂ was 48, 38 and 21% ( P < 0.01) and the cell number per embryo averaged 258, 226 and 188, respectively ( P < 0.01). In Expt 2, the proportion of hatching embryos after 84 h in culture in 1, 5 and 20% O ₂ was 67, 72 and 29% ( P < 0.01), respectively. Cell numbers in the 1 and 5% O ₂ concentrations were higher than in the 20% O ₂ concentration ( P < 0.01). These results indicate that reduction of O ₂ concentration to 5%, well below the frequently used concentration of about 20% O ₂ in 95% air, is beneficial to rabbit embryo development from the zygote to the blastocyst stage. The O ₂ concentration may be more critical with simple defined macromolecule-free media than in media containing serum.