An enzyme from Saccharomyces cerevisiae which catalyzes the reaction: L-galactonolactone + O2 leads to L-ascorbate + H2O2 has been purified 466-fold from the mitochondrial fraction of a yeast homogenate. The enzyme has several properties that are different from the L-galactonolactone oxidase described by Nishikimi et al. [Arch. Biochem. Biophys. 191, 479-486 (1978)]. By gel filtration in the presence of sodium deoxycholate an apparent Mr of 70 000 was obtained for the active enzyme. Polyacrylamide-gradient gel electrophoresis in the presence of deoxycholate gave an Mr of 74 000, whereas sodium dodecylsulfate/polyacrylamide gel electrophoresis showed only one protein band corresponding to an Mr of 18 000. A tetrameric structure of the enzyme is thereby suggested. The substrate specificity is confined to the aldonoacid lactones L-galactono-, D-altrono-, L-fucono-, D-arabino- and D-threono-1,4-lactones. Competitive inhibition was demonstrated with L-gulono- and D-galactono-1,4-lactones. p-Chloromercuriphenyl sulfonate, iodoacetamide, N-ethylmaleimide, sulfite and sulfide were all inhibitory to the enzyme. No effect was seen when cyanide, azide, EDTA, alpha, alpha'-bipyridyl or bathocuproine disulfonate was added. An apparent Km of 0.3 mM with L-galactonolactone as a substrate was found. The Km for oxygen was 0.18 mM. The pH/activity curve exhibited a maximum around pH 8.9 and a shoulder at pH 6.5. Evidence of a covalently bound flavin coenzyme and involvement of an iron-sulfur cluster was obtained from difference spectra of oxidized minus substrate-reduced enzyme with peaks or shoulders of the oxidized enzyme at 475, 445, 410, 375 and 350 nm. In sodium dodecylsulfate/polyacrylamide gels the enzyme subunit(s) had a bright yellow fluorescence after fixation in 7% acetic acid or 5% formaldehyde. The galactonolactone oxidase is stable with 50% activity being lost in 6 months at + 5 degrees C.