Cobalamin metabolism and function were investigated at the levels from transcobalamin II (TCII) receptor to the cobalamin-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase, in a methionine-dependent (P60) and a methionine-independent (P60H) glioma cell line. Using P60H as reference, the P60 cells cultured in a methionine medium had slightly lower TCII receptor activity and normal total cobalamin content, a moderately reduced microsomal and mitochondrial cobalamin(III) reductase activity but only trace amounts of the methylcobalamin and adenosylcobalamin cofactors. When transferred to a homocysteine medium without methionine, P60H cells showed a slightly enhanced TCII receptor activity, but the other cobalamin-related functions were essentially unchanged. In contrast, the methionine-dependent P60 cells responded to homocysteine medium with a nearly 6-fold enhancement of TCII receptor expression and a doubling of both the hydroxycobalamin content and the microsomal reductase activity. The mitochondrial reductase and the cobalamin-related processes further down the pathway did not change markedly. In both cell lines, TCII receptor activity was further increased when growth in homocysteine medium was combined with N2O exposure. These data suggest that low methionine and/or high homocysteine exert a positive feedback control on TCII receptor activity. The concurrent increase in hydroxycobalamin content and in microsomal reductase activity are either subjected to similar regulation or secondary to increased cobalamin transport. This regulatory network is most prominent in the methionine-dependent P60 cells harboring a disruption of the network in the proximity of cobalamin(III) reductase.