Ochratoxin A (OTA), a fungal metabolite produced by several strains of Aspergillus and Penicillium, binds to serum albumin with high affinity only in the completely deprotonated form (dianion). The pK(a) of the phenolic group of OTA decreased by more than three units when it was bound to human serum albumin (HSA). Optical spectroscopy provided evidence that HSA has at least two binding sites for OTA, each being able to accommodate one dianion. These two sites were characterized by the binding constants of 5.2 x 10(6) and 1.0 x 10(5) M-1. The binding constant for the monoanion of OTA was estimated to be similar to10(3) M-1. Fluorescence polarization spectroscopy confirmed weak interaction of the monoanion with the protein in the F and E forms (pH < 4) and showed lower affinity of the dianion to the B form of HSA (pH > 8) compared to the N form (pH similar to 7). Fluorescence anisotropy decay of the dianion of OTA bound to HSA (36.6 ns) was much longer than its emission lifetime (5.2 ns) and was close to reported values for the rotational tumbling time of the HSA molecule. Results of chemical denaturation with 9 M urea or 5 M guanidine hydrochloride established that high-affinity binding of OTA only occurred to the native protein. Efficient energy transfer from the single tryptophan residue of HSA (Trp214) to bound OTA was observed. Analysis of fluorescence data provided an estimate of the distance between the dianion of OTA in its highest-affinity site and the Trp214 residue, which was on the order of 16 Angstrom. The results are discussed with respect to recent crystallographic data for HSA-ligand complexes and pH-dependent conformation of HSA.