Intracellular metabolism of methotrexate (MTX) to MTX-polyglutamates (MTXPG) is one determinant of cytotoxicity. Steady-state accumulation of MTXPG seems to depend on the activity of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamate residues, and gamma-glutamyl hydrolase (GGH), which removes them. Overexpression of GGH would be expected to decrease intracellular MTXPG, thereby increasing efflux of MTX and decreasing cytotoxicity. Increased expression of GGH has been shown to be associated with resistance to MTX in human sarcoma cell lines and a rat hepatoma cell line. To clarify the specific role of GGH in determining MTX sensitivity, we investigated the phenotype produced by forced GGH overexpression in two cell types. Furthermore, because MTX and folic acid share metabolic pathways, we measured the effects of GGH overexpression on folic acid metabolism. The full-length cDNA for GGH, subcloned into a constitutive expression vector, was transfected into a human fibrosarcoma (HT-1080) and a human breast carcinoma (MCF-7) cell line. Compared with the clones containing an empty vector, the GGH-overexpressing cells express 15- to 30-fold more GGH mRNA, more GGH protein, and 15- to 90-fold more GGH enzyme activity. GGH overexpression altered MTX accumulation and metabolism to long-chain polyglutamates. In contrast to expectations, however, GGH overexpression did not confer resistance to short MTX exposures in either cell line. Changes in MTX metabolism were found to be balanced by alterations in accumulation and metabolism of folic acid. The ratio of MTX:folate accumulation may be a better predictor of MTX cytotoxicity than the accumulation of either alone. We conclude that, at least for these two cell lines, GGH overexpression alone is insufficient to produce clinical resistance to MTX.