Racemic amino acid esters were optically resolved via hydrolysis in organic solvents by the catalysis of an industrial alkaline protease, ’Alcalase’. The products which were composed mainly of L-amino acids were insoluble and easily separated by filtration. The activity of the enzyme and enantiomeric excess of the products were significantly dependent on the nature of solvent and the water content in the reaction media. Generally, high values of enantiomeric excess were obtained at low water contents. Many natural and unnatural amino acids were resolved by this method.