The influence of various fatty acids of 14-18 carbon chain lengths on the binding of calcium to human serum albumin was studied. The cis-unsaturated fatty acids (myristoleic, palmitoleic, oleic, and linoleic) caused a much larger increase in the affinity and maximum binding of calcium for albumin than did saturated fatty acids (myristic, palmitic, and stearic). For the unsaturated fatty acids, the influence on calcium-albumin binding increased as the degree of unsaturation increased or the chain length decreased. With oleic or linoleic acid, increases in calcium binding were observed at approximately 2-3 mol of fatty acid/mol of albumin, while for myristic, palmitic, or elaidic acid, concentrations of 5-7 fatty acids/mol of albumin were required. The maximum fluorescence emission of albumin was found to change in parallel with the calcium binding for oleic, palmitic, and elaidic acids. We conclude that 1) cis-unsaturated and saturated or trans-unsaturated fatty acids have very different effects on the binding of calcium to human albumin, 2) the alteration in calcium binding is likely due to a conformational change in the albumin molecule, and 3) changes in calcium binding to albumin are observed at levels of cis-unsaturated fatty acids which are obtainable in vivo.