We developed a method based on the incorporation of 13C2-units derived from [U-13C]glycine that allows the simultaneous quantification of tissue protein and RNA synthesis in vivo. Two groups of 26 mice were fed diets containing a high (HF diet) or a low quantity of fiber (LF diet). After 6 d, [U13C]glycine was added to the diet and groups of four mice were killed after 2, 4, 6, 8, 12 and 24 h. Hepatic and intestinal mucosal free and RNA-bound purine nucleosides were extracted and enzymically degraded to allantoin. Allantoin was degraded to glyoxylate, which was then reductively aminated to glycine, which contains the two 13C-atoms incorporated via de novo synthesis. Ingestion of the HF diet was associated with significantly (P < 0.05) higher rates of total RNA synthesis in both the liver ( HF diet, 29%/d; LF diet, 21%/d) and mucosa (HF diet, 27%/d; LF diet, 17 %/d). The mean rates of RNA synthesis in each tissue were significantly (P < 0.01) lower than the respective rates of protein synthesis (liver, 67%/d; mucosa, 74%/d). The isotopic enrichment of the free purine nucleotide pool increased rapidly and exponentially, but the steady-state value was substantially (P < 0. 001) lower than that of the RNA-bound purines. The results suggest that the free nucleotide pool consists of two kinetically distinct compartments, one of which is small and has a rapid rate of turnover. This, we propose, acts as the RNA precursor pool. The other is large, has a low rate of turnover and, we believe, is the pool of adenosine triphosphate involved in cellular energetics.