In the separation of cobalamins by micro-high-performance liquid chromatography (micro-HPLC), each cobalamin was detected first by UV absorption and then by microbiological method in order to improve the sensitivity of the system. For the elution of cobalamins, methanol concentration in 0.05 M sodium acetate buffer, pH 4.0 as the mobile phase was changed stepwise and cobalamins were eluted in the order of OH-B_<12>, HSO_3-B_<12>, CN-B_<12>, AdoB_<12> and CH_3-B_<12>. By this micro-HPLC method combined with microbiological determination, the sensitivity increased about a hundred times in comparison with that already reported with regard to HPLC.