Schizosaccharomyces pombe has an open reading frame, which we named alr1⁺, encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1⁺ gene in Escherichia coli and purified the gene product (Alr1p), with an M_r of 41,590, to homogeneity. Alr1p contains pyridoxal 5'-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent K_m and V_max values as follows: for L-alanine, 5.0 mM and 670 µmol/min/mg, respectively, and for D-alanine, 2.4 mM and 350 µmol/min/mg, respectively. The enzyme is almost specific to alanine, but L-serine and L-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of L-alanine, respectively. S. pombe uses D-alanine as a sole nitrogen source, but deletion of the alr1⁺ gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for L-alanine coupled with racemization plays a major role in the catabolism of D-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses L-alanine but not D-alanine as a sole nitrogen source. Moreover, D-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1⁺ gene enabled S. cerevisiae to grow efficiently on D-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of D-alanine.