Flavin-linked sulfhydryl oxidases participate in the net generation of disulfide bonds during oxidative protein folding in the endoplasmic reticulum. Members of the Quiescin-sulfhydryl oxidase (QSOX) family catalyze the facile direct introduction of disulfide bonds into unfolded reduced proteins with the reduction of molecular oxygen to generate hydrogen peroxide. Current progress in dissecting the mechanism of QSOX enzymes is reviewed, with emphasis on the CxxC motifs in the thioredoxin and Erv/ALR domains and the involvement of the flavin prosthetic group. The tissue distribution and intra- and extracellular location of QSOX enzymes are discussed, and suggestions for the physiological role of these enzymes are presented. The review compares the substrate specificity and catalytic efficiency of the QSOX enzymes with members of the Ero1 family of flavin-dependent sulfhydryl oxidases: enzymes believed to play key roles in disulfide generation in yeast and higher eukaryotes. Finally, limitations of our current understanding of disulfide generation in metazoans are identified and questions posed for the future.