The effect of low concentrations of inorganic phosphate (P_i) on development, metabolic activity, and mitochondrial organization in the same cohorts of cultured hamster embryos was evaluated. Two-cell embryos were collected from eCG-stimulated golden hamsters and cultured in HECM-10 with 0.0 (control), 1.25, 2.5, or 5.0 μM KH₂PO₄. Glucose utilization through the Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA)-cycle activity were determined following 5 h of culture. Mitochondrial organization in living embryos was evaluated using multiphoton microscopy at 6 h of culture. Development was assessed at 27 h (on-time 8-cell stage) and 51 h (on-time blastocyst stage) of culture. Total cell numbers, as well as cell allocation to the trophectoderm and inner cell mass were determined for morula- and blastocyst-stage embryos. Culture with P_i did not alter TCA-cycle activity. However, culture with ≥2.5 μM P_i significantly increased (P < 0.01) EMP activity compared to control. Mitochondrial organization was significantly (P < 0.01) disrupted by P_i in a dose-dependent manner. Development to the 8-cell, morula/blastocyst, and blastocyst stages was significantly reduced (P < 0.05) in the presence of ≥2.5 μM P_i compared to both control and 1.25 μM P_i. This study clearly demonstrates that, for hamster embryos, inclusion of even exceptionally low concentrations of P_i in culture medium dramatically alters embryo physiology. Additionally, although 2-cell embryos can tolerate some structural disruption without concomitant, detrimental effects on development or metabolic activity, metabolic disturbance is associated with decreased developmental competence.