In the last few years many attempts have been made to differentiate more than 20 Bifidobacterium species. It has been recognized that identification of bifidobacterial species is problematic because of phenetic and genetic heterogeneities. In order to contribute to our understanding of Bifidobacterium taxonomy, we studied Bifidobacterium phylogeny by performing both 16S rRNA and 16S to 23S (16S-23S) internally transcribed spacer (ITS) sequence analyses. In this study, we determined 16S rRNA sequences of five Bifidobacterium strains representing four species, and compared them with the sequences available in the GenBank database, and used them to construct a distance tree and for a bootstrap analysis. Moreover, we determined the ITS sequences of 29 bifidobacterial strains representing 18 species and compared these sequences with each other. We constructed a phylogenetic tree based on these sequence data and compared this tree with the tree based on 16S rRNA sequence data. We found that the two trees were similar topologically, suggesting that the two types of molecules provided the same kind of phylogenetic information. However, while 16S rRNA sequences are a good tool to infer interspecific links, the 16S-23S rDNA spacer data allowed us to determine intraspecific relationships. Each of the strains was characterized by its own ITS sequence; hence, 16S-23S rRNA sequences are a good tool for strain identification. Moreover, a comparison of the ITS sequences allowed us to estimate that the maximum level of ITS divergence between strains belonging to the same species was 13%. Our data allowed us to confirm the validity of most of the Bifidobacterium species which we studied and to identify some classification errors. Finally, our results showed that Bifidobacterium strains have no tRNA genes in the 16S-23S spacer region.