The characterization of six bifidobacteria strains used as starters in the manufacture of fermented milk, containing the raffinose family of oligosaccharides (RFOs) as prebiotics, was carried out by applying in vitro methodologies. Selection criteria were the specific growth rate (μ), generation time (Tg), titratable acidity in milk, and enzymatic activities. Strain growth and acidification rate were determined in milk with and without 2% RFOs. Different growth rates, acidification rates and enzymatic patterns were observed among tested bifidobacterial strains. Bifidobacteria with higher levels of α-galactosidase activity showed growth and acidification rate enhancement when RFOs were added compared with lactose as sole carbon source. Based on the results, Bifidobacterium lactis Bb-12 was selected for further investigation as it possessed the enzymes required to enhance growth and metabolic activity in the presence of RFOs from lupins. © 2006 Elsevier Ltd. All rights reserved.