A numerical analysis based on phenotypic characteristics (89 enzymatic tests and 49 carbohydrate acidification tests), in which experimental strips from Biomerieux-API, La Balme les Grottes, France, were used, was performed to characterize 82 new isolates belonging or related to Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve. A total of 72 strains were isolated from child or adult feces, and the other strains were obtained from human vaginas and bronchi. In this study we also included 38 type and reference strains that were representative of all species of the genus Bifidobacterium and 6 strains belonging to the genus Lactobacillus. DNA-DNA relationships between B. longum and B. infantis were determined by using 19 strains related to these species, as determined by the numerical analysis. The degree of DNA binding was determined by the S1 nuclease method. The phenotypic study revealed that there were six main clusters, which were subdivided into nine subclusters. Subcluster Va contained the type strains of B. longum and B. infantis. The DNA-DNA relatedness values of some of the new isolates were very similar to the DNA-DNA relatedness values of the type strain of B. longum. On the basis of these data, it was difficult to isolate B. infantis strains and then to define B. infantis as a single species separated from B. longum. Subclusters IVb to IVf comprised reference strains of B. breve. Cluster III and subcluster Ia were not identified.