Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB)>multi-metal binding site (MBS)>N-terminal binding site (NTS). Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9+/-5, 1.1+/-0.5, and 0.9+/-0.3x10(4)M(-1), respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed.