Young rats were force-fed a complete or threonine-devoid diet for 3 days. On the fourth morning, rats of each group were injected intraperitoneally with cycloheximide (150 mug/100 g body weight) or saline with [6-14C]orotic acid 30 minutes later, 2 hours before killing. Incorporation of [6-14C]orotic acid into hepatic RNA fractions (whole homogenate, postmitochondrial supernatant, microsomes, ribosomes, nuclei and soluble) revealed elevated levels (cpm/mg RNA) in rats force-fed the threoninedevoid diet in comparison to those of rats force-fed the complete diet. However, treatment with cycloheximide decreased the incroporation of [6-14C]orotic acid into the hepatic RNA fractions of the rats force-fed the threonine-devoid diet to levels that were similar to those in rats force-fed the complete diet with or without cycloheximide treatment. Studies dealing with nucleoli isolated by sucrose gradients from livers of control and experimental rats revealed heavier nucleoli and more radioactive labeled RNA in nucleoli ([6-14C]orotic acid administered 30 minutes before killing) of rats force-fed the threonine-devoid diet than in those force-fed the complete diet for 3 days. Treatment with cycloheximide decreased the elevated incorporation in the experimental rats. Also, electron microscopic studies revealed that after cycloheximide treatment, the enlarged hepatic nucleoli of the experimental rats became smaller and returned to a more normal pattern, as found in the control rats. The studies suggest that active hepatic protein synthesis is involved in the increased hepatic RNA synthesis in rats force-fed the threonine-devoid diet and that following inhibition of protein synthesis, as induced by cycloheximide, there is a rapid inhibition of the accelerated hepatic RNA synthesis observed in rats force-fed the threonine-devoid diet with a rapid reversal toward a normal level, i.e. toward that found in control rats.