Arthrobacter oxydans HAP-1 produces a large amount of DL-alanine from glucose via the reductive amination of pyruvate catalyzed by L-alanine dehydrogenase (ALD). This bacterium was found to be capable of growing on either D- or L-alanine as a sole carbon source with lower growth rates than on glucose. The effects of an alanine racemase (AR) inhibitor on the accumulation and/or consumption of D- or L-alanine by the bacterium suggested that D-alanine was formed by the isomerization of L-alanine and degraded through the reverse reaction. D-Alanine non-utilizing mutants were derived from strain HAP-1 by the staining method using the chromogenic agent 2,3,5-triphenyl tetrazolium chloride. The mutants were obtained were of two kinds: ALD-deficient and AR-deficient. The former lost the ability to grow on both D- and L-alanine, indicating that the ALD of strain HAP-1 functions not only in L-alanine synthesis but also in L-alanine degradation. The latter could utilize L-alanine as a carbon source but not D-alanine; furthermore, they required D-alanine for growth in minimum or nutrient medium. A representative AR-deficient mutant, DAN 75, produced 75.6 g of L-alanine per liter with high optical purity (97% e.e.) in a fed-batch cultivation (glucose was fed to 15% in total) using a jar-fermenter. The L-alanine titer was almost equal to the sum of D- and L-alanine produced by the parental strain. The production rate of DAN 75, as well as that of the parental strain HAP-1, slowed down in the late stage of the cultivation. Examination of the influence of exogenously added alanine revealed this reduced production to be due to a deceleration in the glucose consumption rate arising from alanine accumulated in the medium.