The effective production of (S)-(+)-citramalic acid from itaconic acid with an enantiomeric purity of more than 99.9% was successfully achieved using resting cells of a newly isolated strain, Alcaligenes xylosoxydans IL142. The highest conversion activity was obtained with an itaconic acid concentration of 65.0 g.l(-1). After 30 h of reaction, 68.9 g.l(-1) of (S)-(+)-citramalic acid was produced from 65.0 g.l(-1) of itaconic acid. This is equivalent to a molar yield of 93.1%. This production process is of considerable economic significance because only very few by-products were detected. The ATP and CoA requirements for (S)-(+)-citramalic acid formation from itaconic acid were evaluated using crude cell-free enzymes. Addition of succinate enhanced the production of (S)-(+)-citramalic acid in the presence of CoA and ATP. These results suggest the existence of strong citramalyl-CoA transferase activity in the cell.