When lettuces (Lactuca sativa L.) grown for 30 days in hydroponic culture were treated with various concentrations of cyanocobalamin for 24 h, its content in their leaves increased significantly from nondetectable to 164.6 ± 74.7 ng/g fresh weight. This finding indicated that consumption of only two or three of these fresh leaves is sufficient to meet the Recommended Dietary Allowance for adults of 2.4 μg/day. Analyses using a cobalamin-dependent Escherichia coli 215 bioautogram and LC/ESI-MS/MS demonstrated that the cyanocobalamin absorbed from the nutrient solutions by the leaves did not alter any other compounds such as coenzymes and inactive corrinoids. Gel filtration indicated that most (86%) of the cyanocobalamin in the leaves was recovered in the free cyanocobalamin fractions. These results indicated that cyanocobalamin-enriched lettuce leaves would be an excellent source of free cyanocobalamin, particularly for strict vegetarians or elderly people with food-bound cobalamin malabsorption.