An active second dihydrofolate reductase enzyme is not a feature of rat and mouse, but they do have activity in their mitochondria

•Rodents have one functional DHFR gene while humans have two: DHFR & DHFRL1.•Rodents have DHFR activity in their mitochondria due to the cytoplasmic enzyme.•Wild black rats have the coding capacity for a second DHFR enzyme.•Mouse and rat are not suitable models for studying folate reduction in humans.•Understanding the molecular differences between humans and rodents is warranted. The identification of a second functional dihydrofolate reductase enzyme in humans, DHFRL1, led us to consider whether this is also a feature of rodents. We demonstrate that dihydrofolate reductase activity is also a feature of the mitochondria in both rat and mouse but this is not due to a second enzyme. While our phylogenetic analysis revealed that RNA-mediated DHFR duplication events did occur across the mammal tree, the duplicates in brown rat and mouse are likely to be processed pseudogenes. Humans have evolved the need for two separate enzymes while laboratory rats and mice have just one.