Methotrexate polyglutamates are extensively synthesized when cultured hepatocytes and H35 hepatoma cells are exposed to micromolar concentrations of methotrexate. The predominant species found within the cell have from two to four additional gamma-linked glutamate residues. When either cell type containing a mixture of methotrexate and its polyglutamate derivatives is exposed to medium lacking methotrexate, there is a rapid release of methotrexate. This release has a T1/2 of 2 to 4 min and is apparently complete within 30 to 60 min. Methotrexate polyglutamates leave the cells much more slowly and appear to do so by two mechanisms. Although cleavage to methotrexate and subsequent efflux appears to be quantitatively the more important pathway, there is also a slow, finite loss of intact methotrexate polyglutamates from cells which exclude trypan blue. The T1/2 for the loss of methotrexate polyglutamates by both cell types, when placed in medium lacking methotrexate, is approximately 6 to 8 hr. These results, together with those of an earlier study (Galivan, J. (1980) Mol. Pharmacol. 17:105-110), suggest that the polyglutamate derivatives are forms of methotrexate which are as cytotoxic as methotrexate but which offer a potentially greater capacity for cellular destruction because they are retained longer in the tissue.