The capacity of rat intestinal epithelial cells for de novo purine synthesis and reutilization was studied in vitro with isolated cells and in vivo in functionally hepatectomized rats. De novo purine synthesis and purine reutilization were measured as the rate of incorporation of [14C]-glycine or [14C]-adenine, respectively, into the adenine and guanine pools of the intestinal cells. Isolated intestinal epithelial cells incubated with labeled purine or glycine incorporated only labeled adenine. Labeled glycine, guanine, hypoxanthine or xanthine were not incorporated into cellular adenine or guanine. Labeled glycine, given intravenously to functionally hepatectomized rats, was not incorporated into the adenine and guanine of intestinal villus or crypt cells. Labeled glycine was readily incorporated into hepatic adenine and guanine pools of a sham-operated rat. The failure of glycine to be incorporated into intestinal cells in vitro or in vivo demonstrated that these cells lack de novo purine synthesis. Since adenine was readily incorporated into the adenine pool of these cells, we believe that adenine, which is synthesized in the liver or supplied in the diet, was an important precursor for the nucleic acid synthesis in this study.