The influence of bile sensitivity, lactose transport, and acid tolerance of Lactobacillus acidophilus on in vivo digestion of lactose was investigated. Four strains of L. acidophilus exhibiting varied degrees of lactose transport, beta-galactosidase activity, and bile sensitivity were used to prepare unfermented acidophilus milks. Lactose malabsorption was evaluated by measuring breath H2 excretion off 11 lactose maldigesting subjects following ingestion of four acidophilus test milks. Test meals were fed in a randomized double-blind protocol. Consumption of acidophilus milk (2% fat) containing strains B, N1, and E significantly reduced mean total H2 production compared with that of the control reduced-fat (2% fat) milk, but milk containing strain ATCC 4356 did not differ from the control. Acidophilus milk containing L. acidophilus N1 was the most effective of the four acidophilus milks in improving lactose digestion and tolerance. Strain N1 exhibited the lowest beta-galactosidase activity and lactose transport but the greatest bile and acid tolerance of the four strains. The results indicated that bile and acid tolerance may be important factors to consider when L. acidophilus strains are selected for improving lactose digestion and tolerance.