Activation of antimetabolites to metabolites which are retained in cells and mitochondria is catalyzed by the enzyme folylpolyglutamate synthetase (FPGS). Transcription of the FPGS gene in mouse tissues is regulated by the tissue-specific activation of an upstream promoter only in differentiated tissues which serve as specialized organs of folate metabolism, and in all normal and malignant dividing cell populations from a downstream promoter. The recriprical regulation of these two promoters will be studied as will the function of the isoform of FPGS transcribed from the upstream promoter. The intracellular trafficing of FPGS is gaurenteed by the transcription of either upstream or downstream promoter in a manner which produces both a transcript for FPGS which traffices to the cytosol and a second form which traffices to the mitochondrial compartment. The dynamics and functions of this trafficing will be studied. Finally, the role of specific peptides in the enzyme mechanism will be determined.