Alanine racemases are ubiquitous, almost uniquely prokaryotic enzymes catalyzing the racemization between L- and D-alanine. The requirement for D-alanine as a necessary component of the bacterial cell wall makes this class of enzymes a logical target for the development of novel antibiotics. In an effort to better understand the structure and mechanism of these enzymes, we have cloned the two independent alanine racemases from Pseudomonas aeruginosa, an important opportunistic bacterial pathogen of humans and animals. The dadX(PA) and alr(PA) genes have been sequenced, overexpressed, and their activity was demonstrated by complementing D-alanine auxotrophs of Escherichia coli. Both gene products were purified to electrophoretic homogeneity, the enzymes were characterized biochemically, and preliminary crystals were obtained.