A simplified microbiological assay for vitamin B12 estimation, completed on microtitre plates using a colistin sulphate resistant strain of Lactobacillus leichmannii (NCIB 12519), and cryopreserved cultures is described. The new assay correlated well with a more conventional "tube" assay and was not influenced by the presence of antibiotics in serum. Evaluation of assay performance showed excellent interassay and intra-assay precision with quantitative recovery of added cyanocobalamin over a wide range of additions (94.9%-102.1%). The advantages of short incubation time, easy reading, and minimal reagent costs make this assay an attractive option in the routine clinical laboratory and in research.