Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Greater than 70% of homocysteine in circulation is protein-bound. An in vitro model system using human plasma has been developed to study mechanisms of protein-bound homocysteine formation and establish the equilibrium binding capacities of plasma for homocysteine. Addition of homocysteine to plasma caused an initial rapid displacement of cysteine and a subsequent increase in protein-bound homocysteine. This rapid reaction was followed by a slower oxygen-dependent reaction forming additional protein-bound homocysteine. To determine the equilibrium binding capacity of plasma proteins for homocysteine, plasma was treated with 0.5-10 mM DL-homocysteine for 4 h at 37 degreesC under aerobic conditions. Under these conditions the equilibrium binding capacity was 4.88 +/- 0.51 and 4.74 +/- 0.68 mu mol/g protein for male (n = 10) and female (n = 10) donors, respectively. The mechanism of protein-bound homocysteine formation involves both thiol-disulfide exchange and thiol oxidation reactions. We conclude that plasma proteins have a high capacity for binding homocysteine in vitro.