Gel filtration of serum by use of dextran bisacrylamide beads ("Sephacryl S-200," fractionation range: 5000-250 000 daltons), separates the serum proteins into three distinct peaks. We used an eluent containing (per liter) 140 mmol of sodium, 1.10 mmol of calcium, 0.50 mmol of magnesium, and 10 mmol of 2-([2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]amino)ethanesulfonate buffer (pH 7.43 at 37 degrees C) to provide the physiological conditions necessary to maintain the equilibrium of bound calcium in serum. We examined the calcium binding by each of the three protein groups. Calculating the calcium bound per gram of protein in each peak, we found that proteins in the first peak (alpha2-macroglobulin, IgM, haptoglobins) bind about the same amount of calcium per gram as does albumin. The proteins of the second peak (largely IgG and IgA) bind less calcium than does albumin, which is the protein accounting for 90% or more of the third peak. We also were able to calculate intrinsic association constants for calcium/albumin under physiological conditions.