Two pathways for the formation of biosynthetic protein disulfide bonds have been characterized in the endoplasmic reticulum (ER) of eukaryotes. In the major pathway, the membrane-associated flavoprotein Ero1 generates disulfide bonds for transfer to protein disulfide isomerase (PDI), which is responsible for directly introducing disulfide bonds into secretory proteins. In a minor fungal-specific protein oxidation pathway, the membrane-associated flavoprotein Erv2 can catalyze disulfide bond formation via the transfer of oxidizing equivalents to PDI. Genomic sequencing has revealed an abundance of enzymes sharing homology with Ero1, Erv2, or PDI. Herein the authors discuss the functional, mechanistic, and potential structural similarities between these homologs and the core enzymes of the characterized ER oxidation pathways. In addition they speculate about the possible differences between these enzymes that may explain why the cell contains multiple proteins dedicated to a single process. Finally, the eukaryotic ER protein oxidation and reduction pathways are compared to the corresponding prokaryotic periplasmic pathways, to highlight the functional, mechanistic, and structural similarities that exist between the pathways in these two kingdoms despite very low primary sequence homology between the protein and small molecule components.