DHFR2 (dihydrofolate reductase 2) is a gene located on chromosome 3 that functions primarily at the RNA rather than protein level in most adult cells. While DHFR2 was initially believed to mediate mitochondrial dihydrofolate reductase activity based on recombinant enzyme studies, recent proteomic evidence demonstrates that mitochondrial dihydrofolate reductase activity is actually derived from the DHFR gene (chromosome 3), not DHFR2 1. DHFR2 protein is undetectable in most cell types examined, though a DHFR2-specific peptide was identified in embryonic heart, suggesting developmental-specific expression 1. The primary functional role of DHFR2 appears to be RNA-mediated regulation of one-carbon metabolism. DHFR2 RNA directly influences dihydrofolate reductase activity and DHFR expression levels 2. DHFR2 knockout results in decreased dihydrofolate reductase activity, reduced 10-formyltetrahydrofolate abundance, and downregulation of DHFR mRNA and protein, alongside reduced expression of related one-carbon metabolism enzymes 2. Conversely, DHFR knockdown cells show upregulation of DHFR2 RNA, indicating reciprocal regulation 2. Clinically, DHFR2 has potential relevance to ischemic stroke, where DHFR2 expression was significantly negatively correlated with eosinophil infiltration 3. Additionally, DHFR2 can be targeted alongside DHFR using CRISPR/Cas9 for establishing selection systems in biotechnology applications 4.