This study shows that the intracellular concentration of homocysteine in cultured cells is kept low due to an accumulation in the medium. The intracellular level of homocysteine was decreased when its precursor, methionine, was omitted from the culture medium. Intracellular glutathione and cysteine were lowered in cystine-deficient medium. Intracellular glutathione was also lowered when copper ions were added to the culture medium. It is evident from this study that the intracellular concentration of homocysteine was not influenced by the lowered level of glutathione and/or cysteine. High amounts of homocysteine added to the medium give rise to an increase of intracellular reduced homocysteine, which participates in the transsulfuration pathway and can replace cysteine in the synthesis of gluthathione. The addition of relatively high amounts of reduced homocysteine (500 μmol/l) in the presence of copper ions (100 μmol/l) to the culture medium can be directly toxic to the cells, possibly due to oxygen radicals formed by thiol auto-oxidation. Whilst the level of homocysteine in this study using short-time cell culture experiment is much higher than the mild hyperhomocysteinemia thought to be atherogenic in humans, it is conceivable that over a longer time course these levels of homocysteine could be sufficient to induce endothelial dysfunction, eventually leading to atherosclerosis.