Evidence is presented that alanine racemase activity in E. coli K12 is due to two distinct gene products. The predominant isozyme is inducible by either alanine stereoisomer and repressible by glucose. The gene dadX coding for its structure is located by the dadA gene determining the structure of D-amino acid dehydrogenase. The regulatory site for the expression of both genes, dadR, is located on the other side of dadA. The orientation of the dad operon established by multiple-point crosses and deletion mapping is as follows: fadR ...dadRAX ...hemA. The dadX alanine racemase activity is unusually refractory to changes of incubation temperature. It differs strikingly from that of the other isozyme, probably the product of the alr gene. The latter isozyme shows a typical dependence upon incubation temperature. The synthesis of alr alanine racemase is constitutive in respect of both alanine and glucose. In dadX mutants, in which alanine racemase activity equals only 15% of that in wild-type cells grown in the absence of an inducer or catabolite repressor, the dad operon cannot be induced by D-alanine. We presume, therefore, that L-alanine is involved more directly than D-alanine in dad operon regulation.