The current studies have investigated the role of three disulfide bonds of human transcobalamin II(TC II), a plasma transporter of cobalamin (Cbl: Vitamin B₁₂), on its function and stability. When translated in vitro in the presence or absence of microsomal vesicles, TC II constructs with a single substitution, C3S or C249S, demonstrated synthesis of a stable functional protein. However, TC II synthesized in the presence of microsomal vesicles using constructs with a single (C98S, C147S, C187S, C291S), double (C3/147/S, C98/147/S) or triple (C3/98/147/S) substitution was unstable. In the absence of microsomal vesicles, the percent binding to Cbl-Sepharose matrix by 35[S]-TC II expressed by constructs C3S, C3/147/S, C98/147/S,or C3/98/147/S was 100%, 49%, 52% and 35%,respectively. Upon their reductive alkylation, the binding of TC II expressed by these constructs was reduced to about 25-30%. TC II constructs C3S or C249S, when expressed in TC II-deficient fibroblasts, produced a stable functional protein but those expressed by constructs C147S, C187S, C291S, C3/147/S, C98/147/S, C3/98/147/S were rapidly degraded. The intracellular degradation of TC II expressed by these constructs was inhibited by lactacystin or MG-132 but not by the lysosomal degradation inhibitors ammonium chloride or chloroquin. These studies suggest that optimal binding of Cbl by human TC II is supported by disulfide bonds, C98-C291 and C147-C187 and their disruption results in loss of Cbl binding and their rapid degradation by the proteasomal machinery.