Four hours after either a single injection or continuous infusion of the antitumor agent methotrexate (MTX) plus purified [3'',5'',9(n)-3H]MTX in cynomolgus [Macaca fascicularis] or rhesus [Macaca mulatta] monkeys, 80-98% of the 3H radioactivity present in the plasma was not intact MTX. The percentage of 3H-containing MTX products in the urine after 4 h was considerably less, although more variable. This variability seemed to be related to variability in the amount of the total dose excreted. Non-MTX products were also found in selected tissues and the percentage of intact MTX found 4 h after i.v. injection varied from 2-26%. The percentage of intact MTX was measured by comparing the values obtained using the dihydrofolate reductase assay with values based on the specific activity of [3'',5'',9(n)-3H]MTX. Results obtained by DEAE column chromatography on a few samples showed good agreement with results from the reductase assay. [3'',5'',9(n)-3H]MTX products appeared in peaks eluting from the DEAE column both earlier and later than the MTX peak, with the earlier peaks being present in only small amounts in the urine. After continuous i.v. infusion, only 2% or less of the redioactivity found in the CSF after 4 h represented intact MTX, with the remaining radioactivity eluting earlier than MTX. After direct injection into the left lateral ventricle, all 3H radioactivity in CSF and brain tissue represented intact MTX for up to 4 h after injection. The appearance of MTX products in the plasma and selected tissues of these primates a short time after i.v. injection is compared to other work in experimental animals and man and suggests a greater metabolism of MTX than was previously suspected.