Folylpoly-gamma-glutamate synthetase (FPGS) is essential for mammallian cell survival and is a major determinant of cytotoxicity and selectivity for folate antimetabolites. Here we describe the cloning of a cDNA encoding murine FPGS isolated from L1210 leukemia cells. The amino acid sequence of murine FPGS is 82% identical to human FPGS+ with identical discrete regions of up to 41 residues. Murine FPGS contains two AUG initiation codons, shown to be responsible for mitochondrial and cytosolic forms of the enzyme in human cells  Previous studies indicated species, tissue, and tumor specific differences in mammalian FPGS. The availability of murine FPGS expands the knowledge and understanding of the spectrum of these variations.