Binding equilibria for simultaneous binding of several molecules of two anionic ligands, sulfamethizole/warfarin in one series and sulfamethizole/diflunisal in another, to human serum albumin were studied by equilibrium dialysis. It was found that Klotz's stepwise binding equilibrium concept, extended to cover interaction of two ligands with one carrier, could be used for a quantitative description of binding equilibria. Reciprocity of ligand effects was established at all levels. Heterotropic anticooperativity was present among these pairs of ligands. The experiments were supplemented with observations of albumin binding equilibria for traces of warfarin in the presence of varying amount of oleate, up to 6 mol/mol albumin, by measuring dialysis rates for unbound warfarin. Binding of warfarin to albumin is enhanced upon binding of oleate up to 4 mol/mol albumin, and decreases at higher oleate concentrations. Using stoichiometric (stepwise) binding constants for oleate previously published by Ashbrook et al. [(1975) J. Biol. Chem. 250, 2333-2338], the reverse effect, of warfarin on binding of oleate, was calculated. Simultaneous binding of these ligands to albumin could be described according to the stoichiometric principles as used above for sulfamethizole/warfarin and sulfamethizole/diflunisal.