ortho-Substituted polychlorinated biphenyls (PCBs) make up a large part of the PCB residue found in the environment and human tissues. Our laboratory as well as others have demonstrated that ortho-substituted congeners exhibit important biological activities by aryl hydrocarbon (Ah) receptor-independent mechanisms, including changes in second messenger systems necessary for normal cell function and growth. Previous structure-activity relationship (SAR) studies on second messengers and transthyretin (TTR; prealbumin) binding focused little attention on the ortho-substituted PCBs. Disruption of thyroid hormone (TH) transport is one potentially important mechanism by which PCBs can alter TH homeostasis. A more systematic study of PCB binding to TTR, a major TH transport protein, was undertaken, in which the role of ortho-substitution was more thoroughly investigated. Results from this study indicated that the ortho-only substituted series showed significant binding activity and the relative affinities were 2,2′,6 > 2,2′ = 2,6 ⪢ 2 = 2,2′,6,6′. As anticipated on the basis of steric considerations, bromine was shown to be more active as an ortho-substituent where the relative affinity of 2,2′-Br was equivalent to 2,2′,6-Cl. The congener patterns (di-meta-substitution in one or both rings) most closely resembling the diiodophenolic ring of thyroxine (T4) showed the highest binding activity. Multiple ortho-substituents were shown to decrease binding activity in such patterns. Congener patterns (single meta-substitution in one or both rings) more closely resembling the monoiodophenolic ring of T3 showed significantly lower binding activity, consistent with the relatively low binding activity of T3 and smaller size of chlorine compared to iodine. The addition of ortho-substitution to such patterns gave variable results depending on the substituent relationship (adjacency or nonadjacency) to the pattern. Some patterns such as 2,2′,4,4′,5,5′ showed good binding activity and represent common congeners in the commercial Aroclor mixtures and in the environment. The binding potencies of ortho-PCBs to TTR may represent a signature SAR that predicts specific biologic/toxic effects. In this regard, the binding potencies were consistent with measured biological activities of these PCBs, including effects on cell dopamine content, Ca2+ homeostasis, and protein kinase C translocation in neuronal cells and brain homogenate preparations.