Purified tritium-labeled methotrexate was administered to rats and Sephadex G-15 gel chromatography was used to study the formation of poly-gamma-glutamyl metabolites of methotrexate in different tissues. These metabolities were recognized as tritium-labeled antifolate fractions and were identified by their response to serum pterolypolyglutamate hydrolases and by cochromatography with authentic standards. After doses equivalent to 15 to 20 mg for man, rat liver and kidney were found to contain both 4-amino 10-methylpterolyglutamyl-gamma-glutamic acid and 4-amino-10-methylpterolyglutamyl-gamma-glutamyl-gamma-glutamic acid. With one-third of the dose, only 4-amino 10-methylpteroylglutamyl-gamma-glutamic acid was found. Synthesis of methotrexate polyglutamates in liver and kidney was limited to the interval immediately following methotrexate administration and appeared to occur by sequential addition of single glutamyl residues to so-called "free" intracellular methotrexate. No synthesis of methotrexate polyglutamates was demonstrated in small intestine or thymus. After formation, 4-amino-10-methylpteroyglutamyl-gamma-glutamic acid disappeared from liver and kidney with a half-time of 6.5 days. The effect of these metabolites of methotrexate of cell metabolism is unknown.