The growth of Bifidobacterium breve strain HP2 was completely inhibited by the addition of lactate higher than 4.0％ but not by the addition of acetate. Two kinds of lactate-tolerant mutants were isolated by the nitrosoguanidine treatment, enrichment on a liquid medium with 5％ lactate, and selection on agar plates with 5％ lactate. The mutants were not only able to grow in the presence of 5％ lactate but also improved in viable cell stability in the acidic pH range. In a pH-controlled fermentor, mutant N-1-5 grew at a rate slower than that of the wild type but its growth yield was higher. Notably, mutants were more halotolerant and more osmotolerant than the wild type and they were able to grow in the presence of 3％ NaCl or 25％ lactose at which the wild type entirely stopped the growth. The enzyme activities involved in the lactose metabolism in B. breve were measured to elucidate the biochemical basis for lactate tolerance. In the mutants, activities of several enzymes including phosphoglucomutase decreased compared to the wild-type, which may explain their lower growth rate. However, the activity of lactate dehydrogenase or its nature of inhibition by lactate was not altered.