A previous report (Moore and Bondioli Biol. Reprod. 48:833 1993.) has shown that the addition of high concentrations of the amino acids glycine and alanine to a modified Brinster's mouse ovum culture medium resulted in enhanced development of bovine embryos co-cultured with oviductal cells. This experiment tested whether this effect would be seen with CR1aa (Rosenkrans et al. Biol. Reprod. 49:459 1993) medium and co-culture with Buffalo rat liver (BRL) cells. Oocytes were aspirated from ovaries of slaughtered cows and matured in Ham's F10 medium supplemented with 10% fetal calf serum (FCS) and 0.3 μg/ml LH for 22 to 24 h. Frozen semen was thawed, washed by centrifugation in DPBS and resuspended in 400 μl of fertilization medium containing 50 μg/ml heparin. After 15 min incubation at 38.5°C the sperm suspension was diluted with 10 ml of fertilization medium for a final sperm suspension containing 2 μg of heparin and approximately 1.5 10 6 sperm per ml. Oocytes were inseminated by aspirating the maturation medium and replacing it with 0.5 ml of sperm suspension. At 17 to 18 h after insemination cumulus cells were removed by vortexing and 1-cell embryos assigned to one of two treatments: 1) CR1aa medium (containing 3 mg/ml BSA) supplemented with 10% FCS (control) or 2) control medium containing 10 mM glycine and 1 mM alanine (gly/ala). Embryos were placed in 4-well Nunc plates with 500 μl of culture medium per well and BRL cells at 50-60% confluency. Culture was carried out at 38.5°C under a 5% CO 2 in air atmosphere. Embryos were moved to fresh wells with 50 to 60% confluent BRL cells on Day 3 or 4 and again on Day 6 or 7 of culture with day of insemination Day 0. Expanded blastocysts were counted and removed on Days 7,8 and 9 of culture. The total number of oocytes for control and gly/ala groups were 1452 and 1447, respectively. The proportions of embryos that cleaved and developed to the expanded blastocyst stage between the two treatments were compared by Chi Square analysis. The cleavage rate did not differ between control and gly/ala (77% and 76%, respectively). For the control treatment there were a total of 448 expanded blastocysts (31% of total oocytes and 40% of cleaved oocytes). A higher (P<0.05) proportion of embryos developed to the expanded blastocyst stage in the gly/ala treatment with a total of 529 expanded blastocysts (37% of total oocytes and 48% of cleaved oocytes). There was also a difference (P<0.01) in the number of blastocysts that appeared on Day 7 of culture between the two treatments. Of the 448 total expanded blastocysts in the control treatment, 81 (18%) were present on Day 7. Of the 529 total expanded blastocysts in the gly/ala treatment, 184 (35%) were present on Day 7. In preliminary studies to determine trophoblast and inner cell mass (ICM) cell numbers, 10 expanded blastocysts from the control treatment had a mean of 45±3 trophoblast cells and 18±2 ICM cells. Fifteen expanded blastocysts from the gly/ala treatment had a mean of 56±5 trophoblast cells and 17±1 ICM cells. These results extend the earlier observation concerning the beneficial effect of glycine and alanine for co-culture of bovine embryos.