Cellular uptake of cobalamin (Cbl) is mediated by transcobalamin II (TCII), a Cbl binding protein in the plasma. The TCII-Cbl complex binds to a cell surface receptor and is internalized by endocytosis. We have generated monoclonal antibodies (mAbs) to human TCII that can be distinguished into three functional types on the basis of interaction with three different regions of the protein. Type 1: Receptor blocking. This mAb binds holo-TCII and inhibits the cellular uptake of Cbl. Type 2: Cbl blocking. This mAb binds apo-TCII at or near the Cbl binding domain and inhibits the formation of holo-TCII. Type 3: Precipitating. This mAb binds both holo-TCII and apo-TCII but does not interfere with Cbl binding. Whereas type 1 and type 2 mAb, following incubation with TCII-[57Co]Cbl or apo-TCII, respectively, inhibit the uptake of radio-labeled Cbl by K562 cells, type 3 mAb has no such activity with either form of TCII. These properties of type 1 and type 2 mAb that inhibit the cellular uptake of Cbl, may serve to induce rapid Cbl deficiency and provide a model to study the effect of selective Cbl depletion on cell division and differentiation as well as on the pathways dependent on the two Cbl cofactors, methyl-Cbl and 5'-deoxyadenosyl-Cbl.