The toxic and/or beneficial effects of four metabolic regulators on embryo development were evaluated. In-vitro-produced compact morulae were cultured for 3 days in a chemically defined medium + bovine serum albumin (BSA; CDM-2) plus regulators (4991 total embryos). Phenazine ethosulfate (PES), phloretin (PL), pyrroline-5-carboxylate (P5C), and sodium azide (NaN3) were evaluated at four doses each in factorial combinations with four concentrations of glucose: 0, 0.5, 2, and 8 mm. Phenazine ethosulfate at 0.9 microm resulted in poorer development than lower or no PES. Phloretin was, in general, detrimental for embryo development, but most markedly at the highest dose (270 microm). Pyrroline-5-carboxylate had little effect on post-compaction embryos at the doses studied, 9 to 81 microm. Sodium azide at the concentrations used (3, 9, and 27 microm) had little effect on embryo development compared with controls. Concentrations of glucose had little effect on development of embryos. A fifth metabolic regulator, 2,4-dinitrophenol (DNP), was studied at various doses at pre-morula or morula-blastocyst stages cultured in 2 mm glucose. Embryos (2189 total) cultured in 90 microm DNP developed more slowly and were darker than embryos cultured at lower doses. Embryos cultured in 30 microm DNP had a higher blastocyst rate (48.3%) than controls (34.9%). In the last experiment using G1.2/G2.2 media, DNP (30 microm) resulted in a marked decrease in embryonic development when embryos were exposed at the zygote to 8- to 16-cell stages but had little effect when morulae were exposed for 2 days. The dose-response information for these metabolic regulators is crucial for designing future experiments.