Utilization of various carbohydrates viz., glucose, fructose, sucrose, lactose and galactose by Lactobacillus acidophilus strains was investigated in Lactobacillus Selection Broth. Maximum viable counts, acid production and sugar utilization by different test strains were in the order: glucose greater than or equal to fructose greater than sucrose greater than or equal to lactose greater than galactose. The generation time of the tested strains was shorter in glucose medium as compared to sucrose or lactose medium.