Previous studies by us and our colleagues suggested three distinctive deoxyribonucleic acid (DNA) homology groups among the bifidobacteria; these were provisionally named “catenulatum,” “dentium,” and “angulatum.” One hundred eighty-four strains isolated from sewage, in addition to many of the strains from the previous study, were investigated and their DNA homology relationships were assessed using 23 reference systems. Strains in the catenulatum group were found not to differ significantly from those of Bifidobacterium adolescentis Reuter in their main physiological characters, such as sugars fermented and temperature, pH, and oxygen relationships; however, their DNA reciprocal homology is only some 50%, their guanine plus cytosine values were 54.7 ± 0.2 and 59.4 ± 0.4 mol %, respectively, and there were some morphological differences between them. The DNA of the dentium group has only about 45% homology with the DNA of B. adolescentis and is even less related to other members of the genus. The dentium strains can also be distinguished from other bifidobacteria by means of their sugar fermentations. The DNA of the angulatum group has little or no homology with that of any other bifidobacteria; the angulatum group also has a distinctive pattern of sugar fermentation and a unique morphology, resembling that of the coryneform bacteria. The three groups are named and described as new species of the genus Bifidobacterium: B. catenulatum, B. dentium, and B. angulatum. The type strains of these species are B669 (= ATCC 27539), B764 (= ATCC 27534), and B677 (= ATCC 27535), respectively. DNA-DNA homology relationships are basic to currently proposed species concepts, and data are presented confirming the reliability of critical experimental parameters influencing filter-bound DNA and thus the final relative homology values (e.g., temperature and time of incubation and annealing of DNA in the presence of homologous and heterologous competitive or nonspecific DNA, and the replicability of homology values using different homologous DNA preparations with single DNA competitor and reference DNA).