A new strain, Y-134, which was isolated as a producer of cholesterol oxidase (CHO) with high stability in detergents, produced two cholesterol oxidases; one (CHO-A) adsorbed to DEAE-Sepharose, while the other (CHO-U) did not. Specific properties of purified CHO-U were compared with those of CHO-A [Isobe et al., J. Biosci. Bioeng., 95, 257-263 (2003)]. The amino acid sequences of 30 residues from the NH2 terminus were identical in both enzymes, except for one unascertained residue. CHO-U was also stable in nonionic detergents. However, many other properties of CHO-U were different from those of CHO-A; The purified CHO-U exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 276, 350, and 450 nm, and the enzyme activity was not enhanced by metals. CHO-U was a monomeric enzyme with 58 kDa of molecular mass and pI of 7.0. The optimum pH of CHO-U was more than 0.5 acidic pH in relation to that of CHO-A, but the stability of alkaline pH was higher than CHO-A. In addition, the K-m value for cholesterol was lower than that of CHO-A, and the V-max value was higher.