Results of recent studies showed that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are equipotent in domestic chicken (Gallus gallus domesticus) while PeCDF is more potent than TCDD in ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica). To elucidate the mechanism(s) underlying these differences in relative potency of PeCDF among avian species, we tested the hypothesis that this is due to species-specific differential binding affinity of PeCDF to the aryl hydrocarbon receptor 1 (AHR1). Here, we modified a cell-based binding assay that allowed us to measure the binding affinity of dioxin-like compounds (DLCs) to avian AHR1 expressed in COS-7 (fibroblast-like cells). The results of the binding assay show that PeCDF and TCDD bind with equal affinity to chicken AHR1, but PeCDF binds with greater affinity than TCDD to pheasant (3-fold) and Japanese quail (5-fold) AHR1. The current report introduces a COS-7 whole-cell binding assay and provides a mechanistic explanation for differential relative potencies of PeCDF among species of birds.