The objective was to compare effects of three metabolic regulators on development of post-compaction bovine embryos. In-vitro-produced 8- to 16-cell embryos were allocated to treatments for 72 h in G2.2 medium as follows: 0.3 microm phenazine ethosulfate (PES); 27 microm sodium azide (NaN3); 30 microm 2,4-dinitrophenol (DNP); and control, no regulator. Treatments responded similarly for blastocyst rates and embryo quality responses (P > 0.1). The PES treatment resulted in higher glucose metabolism than the NaN3 treatment (18.5 v. 14.5 pmol per embryo per h, P < 0.05), and both did not differ from DNP or the control. The PES treatment tended to result in more flux of glucose through the pentose phosphate pathway (PPP) than the control (50.5 v. 21.5%, P < 0.11). The NaN3 treatment caused more glucose uptake than the PES treatment (38.9 v. 13.1 pmol per embryo per h, P < 0.01), but neither differed from the control or DNP treatment (P > 0.1). Glycolysis for the PES treatment was 187%, which was higher than any of the other groups (88-94%; P < 0.01). There were fewer medium + large lipid granules in the cytoplasm of PES-treated embryos than any other group, including the in vitro control (P < 0.01). However, in vivo control embryos had still fewer large and medium-sized lipid granules (P < 0.01) than the PES treatment. Developmental competence to Day 14 after embryo transfer was similar among treatments. The PES treatment increased glucose metabolism, tended to increase the PPP flux of glucose and clearly reduced accumulation of lipids in embryos produced in the chemically defined media used. Use of PES in culture media may be a promising approach to improving in vitro production of embryos.