d-isoleucine (d-Ile) can be assayed using chiral chromatography but the availability of that method is limited by the necessity for special expertise and expensive equipment. We therefore developed a simple and specific colorimetric assay system for d-Ile determination using an artificially created NADP(+)-dependent d-amino acid dehydrogenase (DAADH). The system consists of two reaction steps: the first is the quantitative conversion of d-Ile to (3R)-2-oxo-3-methyl valerate by DAADH in which NADP(+) is converted to NADPH, while the second is chemical conversion of NADPH to reduced water-soluble Tetrazolium-3 via a redox mediator. d-Ile was determined from 1 to 50 A mu M, and the assay was unaffected by the presence of any of three other isomers (100 A mu M), alcohol and organic acids.