Optical spectroscopy was used to examine the binding of ochratoxin A (OTA) and warfarin (WAR) to human serum albumin (HSA). Both molecules in the deprotonated form showed high affinity binding to HSA. The close proximity of the highest affinity binding site of the OTA dianion to that of the WAR monoanion was suggested by depolarization of WAR emission in ternary mixtures with [OTA]/[WAR] greater than or equal to 3. Fluorescence polarization data also showed that both OTA and WAR simultaneously bind to HSA for 0.1 less than or equal to [OTA]/ [WAR] less than or equal to 1. The failure of WAR to displace OTA under these conditions is in accord with the much smaller binding constant for WAR. In all displacement experiments either the HSA-to-WAR or HSA-to-OTA concentration ratio was kept constant and close to unity. Evidence of energy transfer from electronically excited WAR to OTA when both species are bound to HSA was obtained from fluorescence emission data. The efficiency of this energy transfer provided an estimate of 27 A as the upper limit of the distance between WAR and OTA. WAR bound to HSA strongly quenched the fluorescence of the single tryptophan residue, Trp214. However, the quenching mechanism was different from the energy transfer mechanism observed for quenching of WAR by OTA. The results show that OTA and WAR share a common binding site in subdomain IIA, with OTA having a higher binding affinity. In addition, WAR can occupy another binding site on HSA when OTA is bound. The data suggest that a secondary binding site of OTA is located in domain III, While that of WAR is in domain I.