A mechanism of 2,3-butanediol (ED) stereoisomer formation was examined with respect to the ED cycle. The enzymes acetylacetoin synthase, acetylacetoin reductase (AACR), and acetylbutanediol hydrolase (ABDH), which are part of the ED cycle, were found to be present in the cell-free extract of the bacterial strain Bacillus cereus YUF-4. Two kinds of acetylbutanediol (ABD) stereoisomers were produced in the reduction of acetylacetoin (AAC) by AACR, which were identified as having 3R,4R and 3S,4R configurations by NMR spectroscopy and an enzymic method. The two ABD formations were found to be catalyzed independently by two respective enzymes : the former was catalyzed by a NADPH-dependent AACR (3R,4R-ABD forming) and the latter by a NADH-dependent AACR (3S,4R-ABD forming). The 3R,4R-ABD was converted into R,R-BD and the 3S,4R-ABD into R,S-BD by intracellular ABDH. These findings demonstrated the existence of a new ED isomer formation mechanism derived from the ED cycle.