The rate and extent of the formation of methotrexate poly-γ-glutamates has been studied in a human breast cancer cell line (MDA.MB.436). Cells were exposed to medium containing 10-7 M radiolabelled methotrexate (MTX) for various periods. Cell extracts were subjected to gel filtration on Bio-Gel P2 and radioactivity in each fraction determined. This has allowed quantitation of the relative amounts of MTX and its polyglutamate derivatives. It has been proposed that MTX polyglutamates may act as storage forms of the drug which could result in prolonged cytotoxicity. This possibility was investigated by determining the ability of the cell line to retain MTX and its olyglutamate derivatives for periods of up to 48 hr after removal of MTX from the incubation medium. Our results show that the lower mol. wt polyglutamates (< three extra glutamic acid residues) rapidly efflux from the cell, while the higher mol. wt species (> three glutamic acid residues) are extensively retained, having an efflux half-life of approximately 30 hr. It has been shown that insulin may potentiate the cytotoxicity of MTX to breast cancer cells in vitro. Our results indicate that in the MDA.MB.436 cell line the total intracellular drug at the steady state is unaffected by insulin (10-6 M). However insulin does result in a 30% increase in the contribution of the higher mol. wt polyglutamates to the total intracellular drug. Our data therefore suggest that the ability of insulin to potentiate the cytotoxic effects of MTX may be related to the hormone's ability to modulate the synthesis of MTX polyglutamates.