Distribution of a Tween-hydrolyzing esterase in the genus Acidiphilium was investigated using 40 strains. The strains were divided into three groups by the cell growth and productivity of extracellular esterase. Only the strains referred to as A. facilis cluster produced a large amount of the esterase in the culture broth. The esterase was inductively produced by adding polyoxyethylene sorbitan acyl esters or sorbitan acyl esters as carbon source, but not by adding polyoxyethylene alkyl ethers or triacylglycerols. Acidiphilium sp. AIU 409 isolated from a strong acid mine drainage showed maximum esterase productivity in the medium containing 1.0% Tween 80 at pH 3.5. The extracellular and cell-bound esterases from strain AIU 409 hydrolyzed polyoxyethylene sorbitan acyl esters and sorbitan acyl esters, but not triacylglycerols. The esterase produced by strains of A. facilis cluster might be a new lipolytic enzyme.