Ochratoxins are fungal metabolites known to contaminate human and animal feed. Ochratoxin A (OTA) is the most widespread form of the toxins and is believed to be responsible for human renal diseases. For the majority of its lifetime within the body, OTA remains bound to the plasma protein human serum albumin (HSA). In this paper, the binding of three OTA derivatives (ochratoxin B (OTB), ochratoxin hydroquinone (OHQ), and O-methylated OTA (MOA)) to HSA is examined using optical spectroscopy. The binding constants decrease as follows: OTA(2-) (5.2 x 10(6) M-1) > OTB2- (1.8 X 10(6) M-1) > OHQ(2-) similar to OHQ(-) (2.2 x 10(5) M-1) > MOA(-) (3 x 10(4) M-1). Studies of the binding of OTB, OHQ, and MOA to recombinant proteins corresponding to the domains of HSA reveal binding to all domains but with different affinities. Similar to OTA, all derivatives exhibit the largest binding constant for domain 2. These ligands are displaced by 2,3,5-triiodobenzoate (TIB), indicating they share a common binding site and bind to Sudlow Site I within domain 2 of HSA. Derivatives with ionizable phenolic protons exhibit a decreased pK(a) by as much as two units upon interaction with HSA. The magnitude of the change in pK(a) observed upon binding decreases in the order OTA > OTB > OHQ. These data suggest a model in which the monoanions of OTA, OTB, and OHQ undergo deprotonation by an arginine within domain 2 upon binding to HSA. The difference in binding constant for the three dianions studied results from the stabilization of the dianion by the surrounding protein matrix.