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The deoxyribonucleic acid (DNA) relatedness between a newly described, homofermentative species, Lactobacillus amylophilus, and a broad spectrum of homofermentative lactobacilli was determined. A low range of DNA reassociation levels (5 to 33%) was observed between L. amylophilus and those species (L. amylovorus, L. bulgaricus, L. casei, L. coryniformis, L. delbrueckii, L. helveticus, L. salivarius, and L. xylosus) with which it shared one or more phenotypic characteristics (such as the production of mainly l-(+)-lactic acid from glucose, hydrolysis of starch, the sugar fermentation pattern) or which had a guanine plus cytosine content of 45 mol%. A lack of DNA relatedness (6 to 20% reassociation) was noted between L. amylophilus and 10 other homofermentative lactobacilli. A DNA reassociation of 90 to 98% was measured among the tested strains of L. amylophilus. Purified cell walls of L. amylophilus contained alanine, aspartic acid, glutamic acid, and lysine, but no diaminopimelic acid. The low DNA relatedness indicated that the L. amylophilus strains were not highly related genetically to the established homofermentative species and, hence, that they represent strains of a distinct genospecies.