This study aimed to identify the numerically predominant bifidobacterial species in feces and mucosa of healthy Spanish people and to determine their phenotypic and genetic diversity. To this end, both traditional culturing and molecular methods were used. A set of 196 bifidobacterial colonies was identified from the counting plates by sequencing of a stretch of their 16S rRNA gene. Representative isolates were phenotypically characterized by their carbohydrate fermentation profile and genotypically typed by RAPD-PCR. Four 16S rDNA libraries composed of 113 clones from two fecal and two mucosal samples were independently analyzed. Seven bifidobacterial species were identified by culturing, and six by 16S rDNA analysis. Both methodologies showed Bifidobacterium longum and B. pseudocatenulatum to predominate in feces and mucosa, although high interindividual variability was noted. High phenotypic variation was observed in the fermentation profile of different isolates from the same species. RAPD analysis showed that two to five strains made up the subjects' personal bifidobacterial communities. The identification of the dominant bifidobacterial species could be useful for the rational design, use, and evaluation of probiotics in our community.