S)-(+)-Citramalic-acid-producing activity in microorganisms was studied with resting cells in a reaction mixture containing itaconic acid. Itaconic-acid-utilizing bacteria were found to produce (S)-(+)-citramalic acid from itaconic acid. The strain, which showed the best productivity among those studied, was identified taxonomically as Alcaligenes denitrificans strain MCI2775. (S)-(+)-Citramalic acid produced by this strain was present in a 99.9% enantiomeric excess. The culture and reaction conditions for the production were optimized for this strain. Addition of Mn2+, D-pantothenic acid and L-leucine to the culture medium enhanced the (S)-(+)-citramalic acid-producing activity. Under optimal conditions, 27 g (S)-(+)-citramalic acid/l was produced in 30 h. The yield to itaconic acid added was 69.0 mol %.