Folylpolyglutamate synthetase (FPGS) catalyzes the attachment of glutamate residues to the folate molecule in plants. Three isoforms of FPGS have been identified in Arabidopsis and these are localized in the plastid (AtDFB), mitochondria (AtDFC), and cytosol (AtDFD). We recently determined that mutants in the AtDFB (At5G05980) gene disrupt primary root development in Arabidopsis thaliana seedlings. Transient expression of AtDFB-green fluorescent protein (GFP) fusion under the control of the native AtDFB promoter in Nicotiana tabacum leaf epidermal cells verified the plastid localization of AtDFB. Furthermore, low concentrations of methotrexate (MTX), a compound commonly used as a folate antagonist in plant and mammalian cells induced primary root defects in wild type seedlings that were similar to atdfb. In addition, atdfb seedlings were more sensitive to MTX when compared to wild type. Quantitative (q) RT-PCR showed lower transcript levels of the mitochondrial and cytosolic FPGS in roots of 7 day old atdfb seedling suggesting feedback regulation of AtDFB on the expression of other FPGS isoforms during early seedling development. The primary root defects of atdfb, which can be traced in part to altered quiescent center (QC) identity, pave the way for future studies that could link cell type specific folate and FPGS isoform requirements to whole organ development.