Synthesis of poly(gamma-glutamyl) metabolites of many antifolates, such as methotrexate (MTX), by folylpolyglutamate synthetase (FPGS) is often essential to their cytotoxic activity. FPGS expression in the MTX-sensitive human T-lymphoblastic leukemia cell line CCRF-CEM and a number of MTX-resistant sublines was previously investigated at the DNA, RNA, and activity levels. Using an FPGS peptide deduced from its cDNA sequence, a rabbit polyclonal antibody to FPGS has now been elicited, immunoaffinity purified, and used to quantitate FPGS protein expression by chemiluminescent Western immunoblot analysis. The antibody was used to determine the half-life of human FPGS protein (3.7 +/- 1.1 h) in parental CCRF-CEM cells. A subline resistant to MTX as a result of amplified dihydrofolate reductase expression shows no change in FPGS protein or activity relative to CCRF-CEM. An MTX transport-defective line, however, displays both higher FPGS protein and activity levels. For several sublines in which the only apparent mechanism of MTX resistance is decreased FPGS activity, the FPGS protein level is decreased proportionally. However, we previously showed that these sublines have the same gene copy number, restriction map, and mRNA size and levels as the parent. Evidently, in these MTX-resistant sublines the mRNA is poorly translated and/or the protein turns over more rapidly.