A novel Fe + Zn containing oxygenase from Acinetobacter johnsonii catalyses 2,3-cleavage of acetylacetone to acetate and methylglyoxal has been purified. The stoichiometry of reactants and products conforms to a classical dioxygenase. The pure protein is a homotetramer of 64 kD with variable amounts of Fe2+ and Zn2+. Activity of the enzyme is more closely related to the Fe2+ content than to the amount of protein. A purification of acetylacetone 2,3-oxygenase, some of its physical properties, and the preference for some analogous substrates are described. (C) 2002 Elsevier Science (USA). All rights reserved.