Abstract A general procedure is described to determine the common free D-amino acids except D-proline in mixtures that also contain L-amino acids. The system employs exogenous pure bacterial D-amino acid transaminase coupled with 2-oxohexanoate, which accepts the amino group from D-amino acids to form D-norleucine. This amino acid is readily quantified by amino acid analysis since it elutes in a position not occupied by any of the common amino acids. Formation of norleucine denotes the presence of some D-amino acid(s) whose identity can be established by a corresponding decrease in the susceptible amino acid(s) after treatment. The utility of the procedure is demonstrated by determination of the amounts of free D-alanine and free D-glutamate in extracts of Escherichia coli JM-103 grown on minimal medium; D-alanine was the major D-amino acid. By the same principle, 2-oxohexanoate through coupling with endogenous bacterial D-amino acid transaminase is shown to be capable of inhibiting the growth of E. coli by depleting it of the D-alanine and D-glutamate.