The taxonomic standing of Bifidobacterium lactis and Bifidobacterium animalis was investigated using a polyphasic approach. Sixteen representatives of both taxa were found to be phenotypically similar and shared more than 70% DNA-DNA relatedness (76-100%), which reinforces the conclusions of previous studies in which B. lactis and B. animalis were considered to be one single species. However, the results of protein profiling, BOX-PCR fingerprinting, Fluorescent Amplified Fragment Length Polymorphism (FAFLP), and atpD and groEL gene sequence analysis demonstrate that representatives of B. animalis and B. lactis constitute two clearly separated subgroups; this subdivision was also phenotypically supported based on the ability to grow in milk. Given the fact that B. lactis Meile et al. 1997 has to be considered as a junior synonym of B. animalis (Mitsuoka 1969) Scardovi and Trovatelli 1974, our data indicate that the latter species should be split into two new subspecies, i.e. Bifidobacterium animalis subsp. animalis subsp. nov. (type strain R101-8T=LMG 10508T=ATCC 25527T=DSM 20104T=JCM 1190T) and Bifidobacterium animalis subsp. lactis subsp. nov. (type strain UR1T=LMG 18314T=DSM 10140T=JCM 10602T).