5,6-Dihydrouridine (D) is a modified base found abundantly in the D-loops of tRNA from Archaea, Bacteria, and Eukarya. D is thought to be formed post-transcriptionally by the reduction of uridines in tRNA transcripts. Despite its abundance, no enzymes that catalyze D-formation have been identified. Using comparative genomics and computational methods we have identified members of the cluster of orthologous genes, COG0042, as putative dihydrouridine synthase encoding genes. Escherichia coli contains three COG0042 family members (yjbN, yhdG, and yohI). Strains were created where one, two, or all three of the COG0042 genes were deleted. Purified tRNA samples were investigated from the three single and the three double knockout strains, as well as from the triple deletion strain. The results showed that the COG0042 gene family is responsible for tRNA-dihydrouridine synthase activity in E. coli. They also suggest that the COG0042-encoded family members act site-specifically on the tRNA D-loop and contain non-redundant catalytic functions in vivo.