The existence of metabolically distinct pools of S-adenosyl-L-methionine in Saccharomyces cerevisiae and isolated rat hepatocytes was investigated. Utilizing a relatively long labeling period with [methyl-14 C] methionine, a metabolically ‘stable’pool was labeled. A subsequent short labeling with [methyl-3 H] methionine selectively labeled a putative metabolically ‘labile’pool. The existence of these distinguishable pools was ascertained by following the 3 H and 14 C label disappearance in S-adenosyl-L-methionine during the chase-period in label-free media containing cycloleycine to prevent futher synthesis of S-adenosyl-L-methionine. In both yeast and hepatocytes, the 3 H 14 C ratio in S-adenosyl-L-methionine decreased sharply. The individual 3 H and 14 C decrease in S-adenosyl-L-methionine showed t 1 2 values of 3 and 8 min for yeast and 4 and 18 min for hepatocytes. The results strongly indicate that at least two metabolically distinct S-adenosyl-L-methionine pools actually do exist in both systems. Subcellular fractionation revealed that the ‘labile’pool exist in the cytosol for both yeast and hepatocytes while the ‘stable’pool exists in the vacuolar and the mitochondrial fraction for the yeast and hepatocytes respectively. The S-adenosyl-L-methionine pools were also studied in normal yeast under anaerobic chase condition and petite mutant yeast. Sharply contrasting with aerobically chased normal yeast, both showed closely parallel 3 H and 14 C decreases in S-adenosyl-L-methionine.