Cycloheximide (20-200 mg/l) did not affect basal D-[U-14C]xylose uptake by rat soleus muscle (2.4 +/- 0.2 mumol . g-1 . h-1). However, the stimulatory effect of insulin on sugar transport was progressively reduced from 375% above basal in control muscles to 170% in muscles exposed to 200 mg cycloheximide/l but above this concentration cycloheximide inhibited basal xylose uptake without further effect on the incremental effect of insulin. Cycloheximide affected the insulin dose-response curve both by depressing insulin sensitivity and by reducing the maximum stimulatory effect of the hormone. In contrast to the inhibition of insulin action, which increased progressively over the range 20-200 mg cycloheximide/l, muscle protein synthesis was inhibited maximally at a concentration of 10 mg/l. Cycloheximide also inhibited the insulinomimetic effects of anoxia, 2:4-dinitrophenol, salicylate, cooling, hydrogen peroxide, diamide, vitamin K5, hyperosmolarity and EDTA, but did not affect concanavalin A-stimulated xylose uptake. It is concluded that cycloheximide inhibits insulin-stimulated sugar transport at some late post-receptor step, and that this effect of cycloheximide is not secondary to the inhibition of protein synthesis.