Indian River male broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 18, 24 and 30% protein + 0 or 1 mg triiodothyronine (T3)/kg of diet to study energetic costs of lipogenesis and the use of various substrates for in vitro lipogenesis. 2. De novo lipid and CO2 production were determined in the presence of [1-14C]pyruvate, [2-14C]pyruvate, [3-14C]pyruvate, [2-14C]acetate and [U-14C]alanine. 3. Oxygen consumption was determined in mitochondrial preparations to estimate the energetic costs in explants synthesizing lipid. 4. Radiolabeled CO2 derived from [1-14C]pyruvate was used as an estimate of coenzyme A availability in liver explants. Lipids derived from [2-14C]pyruvate, [2-14C]acetate and [U-14C]alanine estimate relative substrate efficiency. 5. Labeled CO2 production from [1-14C]pyruvate was greatest in that group fed a 12% protein diet and least in the group fed a 30% protein diet. 6. In addition, T3 increased CO2 production from [1-14C]pyruvate. 7. The production of 14CO2 from the second carbon of pyruvate or acetate was increased by T3. 8. The low-protein diet (12% protein) increased (P < 0.05) lipogenesis. 9. Adding T3 to the diets decreased carbon flux into lipid from all substrates, but increased CO2 production from all substrates without changing stage 3 and 4 respiration rates in mitochondrial preparations. 10. These observations imply that coenzyme A availability may have regulated de novo lipogenesis in the present study. 11. It was also concluded that previously noted effects of T3 on intermediary metabolism may involve metabolic pathways that do not involve changes in mitochondrial function.