The action of cycloheximide on some parameters of glucose and lipid metabolism was studied in vitro in epidiymal fat pads from fasted rats. Incubation of fat pads with cycloheximide (1 mug/ml) for 2 hours resulted in a two-fold increase in glucose uptake, glucose oxidation, incorporation of glucose into lipids, and reesterification of free fatty acid. The increase in glucose oxidation was evident in experiments in which [U-14C], [1-14C], or [6-14C]glucose was added to the media, but it was absent when the media were supplemented with pyruvate. Incorporation of glucose into glycogen and accumulation of lactate in the medium were not seriously modified by the presence of cycloheximide. The stimulatory effect of cycloheximide on incorporation of glucose into lipids was absent when insulin or cortisol was added to the medium. A cycloheximide-mediated increase in glucose uptake seems to be responsible for the subsequent changes in glucose metabolism, and would seem to be independent of an inhibition in protein synthesis; puromycin and actinomycin D did not mimic the cycloheximide action on glucose incorporation into lipids.