Prebiotics are nondigestible oligosaccharides that selectively stimulate the growth of beneficial bacteria in the human intestine. Fructooligosaccharide (FOS) and inulin are among the most common prebiotics used in food products and dietary supplements. Previous studies examining the bioactivity of lactulose suggest that it also has a prebiotic potential. Lactulose is a derivative of lactose in which the glucose moiety is isomerized to fructose in the presence of heat; and hence can be found in heated milk. The goal of this study was to establish growth kinetics of common probiotics cultured in FOS, inulin or lactulose as the sole carbohydrate source. Fermentation of the prebiotics and the antioxidant activity of spent medium were also characterized. Dextrose and non-probiotic species were included for comparison. Eight commercial and ATCC strains were cultured in a semi-defined modified MRS, modified MRS plus l-cysteine-HCl, or peptone medium containing 1% carbohydrate. When incubated at 37°C in aerobic condition, except for Lactobacillus casei, probiotic strains cultured in prebiotics did not reach maximum growth rate or yield compared with dextrose. Multiple pair-wise comparisons showed that lactulose tended to produce better growth than FOS and inulin in L. rhamnosus and L. acidophilus. In anaerobic condition, non-probiotic species were able to catabolize all carbohydrate sources, but displayed weaker lactic acid production (0.13 ± 0.06%) relative to Bifidobacterium spp. (0.30 ± 0.09%) or Lactobacillus spp. (0.56 ± 0.27%), as measured by titratable acidity. Antioxidant activity, with Trolox as the reference standard, was inversely correlated with the pH of the spent medium after fermentation (P = 0.022). Among the 3 prebiotic substrates, lactulose yielded the lowest pH in L. acidophilus and B. infantis, while FOS appeared to be preferred by L. casei and B. bifidum. Antioxidant activity was overall the highest from lactulose fermentation. In conclusion, lactulose is a promising prebiotic ingredient that can be incorporated in functional food products.