The kinetics for vitamin B12, cobalamin (Cbl), transfer across the human placenta, and the retention of the endogenous Cbl and its release into the maternal and fetal compartments were investigated in a dually perfused human term placenta in vitro. After 4 hours of perfusion following a single bolus injection (peak maternal perfusate 94 fmol/ml) of 57Co-Cbl into the maternal reservoir, the maternal [57Co-Cbl] rapidly decreased whereas the fetal [57Co-Cbl] was only 9% of the final maternal [57Co-Cbl]. Even though there was a limited transfer of 57Co-Cbl to the fetus, the placenta rapidly accumulated Cbl. At 4 hours, 18% of the initial 57Co-Cbl dose was in the placenta; only 3% of the initial dose was in the fetal perfusate. Also after 4 hours, [57Co-Cbl] was bound > 95% to transcobalamin (TC)I/III-like proteins in the maternal perfusate, whereas Cbl was bound to TCI/III- and TCII-like proteins with some free in the fetal perfusate. In the cytosol, > 95% of the [57Co-Cbl] was bound (80% to TCII-like and 19% to TCI/III-like proteins). When no exogenous Cbl was added, total endogenous [Cbl] in the maternal circulation increased with time during 8 hours of perfusion at a rate of 0.25 +/- 0.12 pmol/gm per hour. Only 2% of this Cbl was free, whereas 98% was bound to specific binding proteins. Neither plateau values nor equilibration with the fetal side were noted. In the fetal circulation, there was a release of Cbl at a rate of 0.015 +/- 0.003 pmol/gm per hour, which was 99.99% bound. Thus the human placenta rapidly concentrates Cbl in the perfused lobule with little distribution of Cbl to nonperfused areas. Therefore, the human placenta modulates the asymmetric transfer of vitamin B12 on the basis of release of specific Cbl-binding proteins (TCI/III- and TCII-like) into the maternal and fetal perfusates.