In this paper, we provide a systematic analysis of glyoxal (1) formation from a range of monosaccharides and related compounds, to determine their potential role as sources of this α-oxoaldehyde in vivo. Substrates were reacted with the Fenton reagent (Fe 2+ /EDTA/H 2 O 2 ) and the mixtures were analyzed by HPLC using the 6-hydroxy-2,4,5-triaminopyrimidine fluorimetric assay. The rank order of hexoses and their derivatives as glyoxal sources was found to be fructose>glucose=mannose=galactose>glucose-6-phosphate>mannitol. Within the pentose group, arabinose and ribose gave the higher yields of 1 followed by deoxyribose and its adenine N-glycosides and ribulose. Among the tested substrates, three-carbon compounds, that is, trioses and glycerol, but not glyceraldehyde-3-phosphate, were by far the most effective sources of 1. The effects of H 2 O 2 and Fe 2+ /EDTA concentrations as well as of other metal ions were also investigated.