The ddlA gene from Salmonella typhimurium coding for D-alanine-D-alanine ligase (ADP-forming) has been subcloned behind the tac promotor in the plasmid pKK223-3, with expression in Escherichia coli JM105. The overexpression system yields 58 mg of active enzyme from 12 g of wet cell paste after 40-fold purification to homogeneity. 5,5'-Dithiobis-(2-nitrobenzoic acid) titrations indicate that all four cysteine residues exist as free thiols. Two crystal forms of the 39,300 Mr enzyme have been produced. A tetragonal form grows at 21 degrees C from 10 to 15% (w/v) polyethylene glycol 8000 in space group P4(1)2(1)2, with two molecules in the asymmetric unit; it has cell constants a = b = 83.8 A, c = 220.0 A, and diffracts to 2.9 A. A monoclinic form grows from 30% (w/v) ammonium sulfate in space group P2(1), with two molecules in the asymmetric unit; it has cell constants a = 60.4 A, b = 102.1 A, c = 64.3 A, beta = 115.7 degrees, and diffracts to 2.2 A resolution.