Xanthine dehydrogenase has been purified to a homogeneous state from cell-free extracts of a strain of Streptomyces. The enzyme has a molecular weight of 125, 000 and consists of two subunits with a molecular weight of 67, 000. The isoelectric point is at pH 4.4. The enzyme exhibits absorption maxima at 273, 355, and 457 nm and contains FAD, iron, and labile sulfide in a molar ratio of 1 : 7 : 1 per subunit. Little molybdenum could be detected. The enzyme is most active at pH 8.7 and at 40°C, and is stable between pH 7 and 12 (at 4°C for24 h) and below 55°C (at pH 9 for 10 min). The activity is stimulated by K⁺ at a concentra- tion of 50 mM or more and also by keeping the enzyme at pH 9 to 11. The activity is inhibited by cyanide, Tiron, and p-chloromercuribenzoate and by adenine and urate. Among the compounds tested, hypoxanthine, guanine, xanthine, 2-hydroxypurine, and 6, 8-dihydroxy-purine are oxidized at considerable rates; hypoxanthine is the best substrate. NAD⁺ is the prefered electron acceptor. K_m values of the enzyme for hypoxanthine, guanine, xanthine, and NAD⁺ are 0.055, 0.015, 0.15, and 0.11 mM, respectively. Marked differences in the properties of this enzyme compared to others are the activity towards guanine, which has a higher affinity for the enzyme than hypoxanthine and xanthine, and a higher reactivity with hypoxanthine than xanthine. The organism has been identified as Streptomyces cyanogenus.