The synthesis of methotrexate poly-γ-glutamates by the MDA-MB-436 and MCF-7 human breast cancer cell lines is highly dependent on the rate of cell growth. Slowly proliferating cells accumulate methotrexate to the same extent as rapidly proliferating cells but convert a lower percentage of the drug to polyglutamate forms. The longest polyglutamate-derivatives of methotrexate are generally only synthesized when the cells are doubling rapidly. The MDA-MB-436 cells exhibit a biphasic response of doubling time and polyglutamation to increasing initial cell number. Extremes of cell density are associated with long doubling times and reduced polyglutamate synthesis. MCF-7 cells show increasing doubling time and decreasing polyglutamate synthesis in response to increasing initial cell number.