Guinea pig aldehyde oxidase was purified about 120-fold at a yield of 26% from liver cytosol by sequential column chromatography using DEAE-cellulose, FMN-Sepharose 4B, and Sephacryl S-300. The purified enzyme showed many similarities with the rabbit liver aldehyde oxidase reported by other workers with respect to its absolute spectra, molecular weight, and cofactor compositions of molybdenum, FAD, and nonheme iron. This enzyme efficiently utilized 2-hydroxypyrimidine and benzaldehyde as electron donors while N1-methylnicotinamide was 40 times less effective than 2-hydroxypyrimidine. Diphenyl sulfoxide was reduced anaerobically to diphenyl sulfide in the presence of electron donors. This activity was highly susceptible to SKF 525-A as well as the known inhibitors for aldehyde oxidase such as menadione, estradiol, and potassium cyanide. This enzyme also reduced dibenzyl sulfoxide, phenothiazine sulfoxide, D-biotin methyl ester d-sulfoxide, and quinoline N-oxide, but not L-methionine sulfoxide, dimethyl sulfoxide, D-biotin methyl ester l-sulfoxide, and D-biotin d- and l-sulfoxides, as well as diphenyl sulfone. These results indicate that aldehyde oxidase in guinea pig liver functions as a sulfoxide reductase with selective substrate specificity under anaerobic conditions.