Bisphenol A [2,2-bis (4-hydroxyphenyl) propane, BPA] is a versatile industrial material for plastic products but increasingly being recognized as a pervasive industrial pollutant as well. Accumulating evidence indicates that the environmental contaminant BPA is one of the endocrine disrupting chemicals (EDCs) that can potentially affect humans as well as wildlife adversely. To define the molecular aspects of BPA action, we first investigated the molecules with which it physically interacts. High BPA-binding activity was detected in the P2 membrane fraction prepared from rat brains. As determined by SDS-PAGE analysis, the molecular weight of a BPA-binding protein purified from the rat brain P2 fraction was 53 kDa. The N terminal amino acid sequence of the purified BPA-binding protein was identical to that of the rat protein disulfide isomerase (PDI), which is a multifunctional protein that is critically involved in the folding, assembly, and shedding of many cellular proteins via its isomerase activity, in addition to being considered to function as an intracellular hormone reservoir. The K_d value of BPA binding to recombinant rat PDI was 22.6 ± 6.6 µM. Importantly, the binding activity of 3,5,3'-triiodo-L-thyronine and 17-estradiol hormones to PDI was competitively inhibited by BPA, in addition to abolishing its isomerase activities. Herein, we report that the ubiquitous and multifunctional protein PDI is a target of BPA and propose that the binding to PDI and the subsequent inhibition of PDI activities might be mechanistically responsible for various BPA actions.