An efficient process of optical resolution is provided for producing an L-amino acid represented by the following formula (I): ##STR1## wherein R is --CH.sub.2 CO.sub.2 H, --CH.sub.2 CONH.sub.2, --CO.sub.2 H or ##STR2## in which R.sup.1 is hydrogen atom, an alkyl group having 1 to 10 carbon atoms or a halogen-substituted alkyl group having 1 to 10 carbon atoms. The process comprises an optical resolution of an N-substituted carbonyl-D,L-amino acid represented by the formula (II): ##STR3## (wherein R is the same as defined above and R.sup.2 is an alkanoyl group having 1 to 5 carbon atoms, benzoyl group, a halogen-substituted alkanoyl group having 1 to 5 carbon atoms or a halogen-substituted benzoyl group), its salt or a mixture thereof, by means of cells of a microorganism, a fermentation broth of a microorganism, a treated fermentation broth of a microorganism or a mixture thereof, said microorganism being selected from the bacteria of the genera Serratia, Staphylococcus, Bacillus, Flavobacterium, Achromobacter and Alcaligenes and actinomycetes of the genera Actinoplanes, Streptosporangium and Sebekia.