Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined medium (modified Tyrode's solution) without glucose. When different concentrations of NaCl were added to the medium, the proportions of embryos developed to the >or=8-cell, morula and blastocyst stages 96, 144 and 192 h post insemination, respectively, were significantly higher at 89 to 114 mM than 64 to 76 and 126 to 139 mM NaCl. A high proportion (28%) of blastocyst-stage embryos 192 h post insemination was obtained at 89 mM NaCl. When calculated osmolarity in the medium with 64 mM NaCl was varied by adding D-sorbitol, significantly higher proportions of morula-stage embryos were obtained at 265 to 315 mOsm (27 to 38%) than 215 (9%) and 365 (2%) mOsm, but the development to the blastocyst stage was difficult at any osmolarities (215 to 365 mOsm) tested. In the medium with a fixed osmolarity (315 mOsm) but with different concentrations (64 to 114 mM) of NaCl, there were no differences in the proportions (29 to 33%) of morula-stage embryos among different NaCl concentrations. However, significantly higher proportions of embryos developed to the blastocyst stage at 89 to 101 mM (22 to 23%) than 64 to 76 (0 to 9%) and 114 (11%) mM NaCl. When Cl- concentration in the medium with 64 mM NaCl was adjusted by adding choline chloride, significantly higher proportions of embryos developed to the morula stage at 97 to 122 mM (32 to 40%) than 72 (6%) and 147 (2%) mM Cl-, but few embryos developed to the blastocyst stage at any Cl- concentrations (72 to 147 mM) tested. In the medium with 64 or 114 mM NaCl and each with 2 different Na+/K+ ratios, there were no differences in the proportions of morula- and blastocyst-stage embryos between different Na+/K+ ratios (31 and 39 at 64 mM NaCl, and 39 and 47 at 114 mM NaCl) at each NaCl concentration. When glucose was added to the medium with 89 mM NaCl 120 h post insemination, there were no significant differences in the proportions (40 to 48%) of morula-stage embryos 144 h post insemination among different concentrations (0 to 6.95 mM) of glucose. The proportion (33%) of blastocysts 192 h post insemination at 2.78 mM glucose was significantly higher than the values at 0 (22%), 5.56 (19%) and 6.95 (15%) mM but not different compared with the values at 1.39 (23%) and 4.17 (28%) mM. In conclusion, NaCl concentration in a defined medium is one of the most important factors for the development of bovine embryo to the blastocyst stage, but the development of embryos up to the morula stage is also regulated by osmolarity and/or Cl-concentration.