Studies were carried out to determine the precursor of the nitrogen-containing ring of rapamycin. The addition of l -lysine and d,l -pipecolate to the medium of Streptomyces hygroscopicus did not affect the specific production of rapamycin. In a basal medium containing no l -lysine except for that in the yeast extract component, l - 14 C-lysine and d,l - 3 H-pipecolate were incorporated to a high degree into rapamycin, d,l - 3 H-pipecolate being incorporated at a higher efficiency. Unlabeled pipecolate reduced the incorporation of radioactive lysine much more than unlabeled lysine decreased the incorporation of radioactive pipecolate. These results indicate that pipecolate is the more direct precursor of rapamycin, lysine being converted to pipecolate and free pipecolate being incorporated directly into rapamycin. Rapamycin samples enriched with 14 C-lysine and 3 H-pipecolate were hydrolyzed and analyzed to provide evidence of specific incorporation of these precursors into the pipecolate moiety .