The gene (choA(A)), encoding cholesterol oxidase from Arthrobacter simplex F2, was cloned and sequenced by polymerase chain reaction. The gene consists of 1653 base pairs and encodes a protein of 551 amino acids (aa). N-terminal sequence analysis of the extracellular cholesterol oxidase of A. simplex F2 confirmed that the mature enzyme consists of 502 aa with a predicted molecular mass of 54,269 Da, and is translated with a 49 aa signal sequence. The structure gene for ChoA(A) was cloned and expressed efficiently in Escherichia coli and Pichia pastoris. The deletion of the choA(A) signal sequence was favorable for the expression of extracellular cholesterol oxidase by P pastoris. (c) 2006 Elsevier Inc. All rights reserved.