alpha-Glycerophosphate oxidase, in a strain of Streptococcus faecium, was found to be adaptive to aerated conditions of growth. The enzyme was purified and found to mediate electron transfer from alpha-glycerophosphate to O(2), with the production of stoichiometric concentrations of H(2)O(2) and dihydroxyacetone phosphate. The enzyme is an anionic flavoprotein, with flavine adenine dinucleotide as the apparent prosthetic group. By manometric methods, a K(m) of 6 x 10(-3)m, with reference to substrate concentration, was obtained. An active reduced nicotinamide adenine dinucleotide diaphorase was closely associated with this enzyme in chromatographic mobility on ECTEOLA-cellulose. The purified alpha-glycerophosphate oxidase was not inhibited by KCN, azide, or sulfhydryl reagents, nor was it stimulated by alpha-lipoate, yeast extract, or other supplements.