The novel bacterium, Rhodococcus sp. PKPD-CL was isolated and identified from the 'Chilika Lake' located at Odisha state of India, which is a largest brackish water habitat in Asia. Rhodococcus sp. PKPD-CL produces extracellular halo tolerant, detergent and organic solvent stable alkaline cholesterol oxidase. It has apparent molecular weight of 60 kDa and was purified 59 fold by using 60% saturated ammonium sulfate fractionation, anion exchange followed by size exclusion chromatographic techniques with 37% recovery. It showed substrate specificity for 3 beta-hydroxysteroids with K-m of 1.1 x 10(-4) M for cholesterol. The pH, 8.0 and the temperature, 37 degrees C were required for its optimum activity. Enzyme is considerably stable at pH 6.0-8.5 and temperature up to 50 degrees C. At 4 and 30 degrees C it maintained its 100% activity up to 60 days. The isoelectric point of the enzyme was 9.5. It showed 80% residual activity with 20% NaCl (3.42 M) and 83% relative activity with 12% NaCl (2.05 M) concentration. The metal ions like Zn2+, Cu2+, Ag+, Fe3+, Ba2+ inhibited the enzyme activity >60% while Hg2+ served a potent inhibitor whereas Mg2+ found to be a good enhancer for it. The enzyme was stable in presence of chemical reagents (NaN3, EDTA), detergents (Tween-80, Tween-20, Triton X-100, sodium cholate) and various organic solvents (isopropanol, ethanol, benzene, chloroform, methanol, toluene, ethyl acetate, butanol and dimethylsulfoxide). Such a multi stress tolerant and versatile enzyme produced by Rhodococcus sp. PKPD-CL may serve as a good choice for industrial applications. (C) 2015 Elsevier Inc. All rights reserved.