Studies were carried out on the effect of triiodothyronine (T3) on the oxygen consumption of dispersed rat liver cells incubated for 2 hr at 37 degrees C. Thyroidectomized SD-NIH rats were kept on a low iodine diet with calcium chloride in the drinking water for 4 weeks or longer to assure hypothyroidism, verified by low serum thyroxine and T3 concentrations. Liver cells were obtained by portal vein perfusion with oxygenated collagenase-enriched Krebs-Ringer-bicarbonate buffer, after the method of Berry and Friend. Cell viability was evaluated by morphology, by trypan blue exclusion, and by biochemical parameters prior to 2-hr incubations with or without added hormone. The oxygen consumption of cell suspensions was measured with the Clark oxygen electrode after the 2-hr incubations at 37 degrees C with oxygenation of the flasks and alanine (5-10 mM) as substrate. In 31 experiments the oxygen consumption (QO2) was enhanced to 121% of control values with T3 in the medium at 3.3 nM ("physiological" level) and with an even greater effect (138% of control values) with 300-1000 nM T3 ("hyperthyroid" level). Cycloheximide at 100 microM was used to inhibit new protein synthesis by incubated hepatocytes. In 18 parallel experiments with cycloheximide blockade, no alteration of the stimulatory effect of T3 was evident. The results signify that incubated liver cells show an early response to thyroid hormone by extranuclear pathways that do not require new protein synthesis.