One hundred and six isolates of the genus Bifidobacterium, isolated from different environments (mainly gastrointestinal), were identified and classified taxonomically to species level by amplified ribosomal DNA restriction analysis. Two restriction endonucleases (Sau3AI and BamHI) were chosen for aligning the 16S rRNA sequences of 16 bifidobacterial species retrieved from various databases, to obtain species-specific restriction patterns. A rapid and accurate identification scheme was obtained by comparing the resulting 16S rDNA digestion profiles of 16 Bifidobacterium type-strains and 90 strains of various origins. All of the investigated strains were previously confirmed at the species level as belonging to the genus Bifidobacterium by fluorescence in-situ hybridisation and by polymerase chain reaction amplification with genus- and species-specific primers. The present work demonstrates that species-specific detection of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium coryneforme, Bifidobacterium cuniculi, Bifidobacterium dentium, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium suis, Bifidobacterium magnum, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum and Bifidobacterium pullorum present in different micro-ecological environments (e.g. gastrointestinal tract) can be accomplished in a reliable, rapid and accurate manner, circumventing the recognised deficiencies of traditional identification techniques.