A series of polychlorinated hydroxybiphenyls (PCBs) has been tested for their binding activity to soluble uterine estrogen receptor protein. Competitive binding analysis was performed on 0-40% ammonium sulfate-enriched uterine cytosol receptor preparations which improved the binding activity for the PCB compounds by a factor of 10-40, by decreasing the nonspecific binding. The binding activities have been correlated to molecular properties supported by molecular modeling studies which emphasize the importance of conformational restriction. The estrogen receptor bound 4-hydroxy-2',4',6'-trichlorobiphenyl (4H2',4',6'TCB) with the greatest affinity, with the concentration of unlabeled inhibitor yielding half-maximal specific binding relative to estradiol (C50) being approximately 42 compared to estradiol, C50 approximately 1.0. PCB compounds that demonstrated appreciable receptor-binding activity were also active in vivo in stimulating uterine weight increases, whereas weak binders were inactive. The 4H2',4',6'TCB compound represents a high degree of conformational restriction around the interring bond due to the presence of two ortho-chlorine atoms. The other PCBs in this series, which show lower receptor-binding activity, vary in position of chlorine substituents and can assume multiple low energy conformations as a result of less hindrance to rotation around the interring bond.