Determining the effects of complex mixtures of environmental contaminants poses many challenges within the field of ecotoxicology. In this study, graded concentrations of herring gull egg extracts, collected from five Great Lakes breeding colonies with variable burdens of organohalogen contaminants (OHCs), were administered to chicken embryonic hepatocytes to determine effects on 7-ethoxyresorufin-O-deethylase (EROD) activity, porphyrin accumulation, and mRNA expression. EROD activity and porphyrin accumulation permitted the ranking of colonies based on the efficacy of eliciting an aryl hydrocarbon receptor-mediated response. An avian ToxChip polymerase chain reaction (PCR) array provided more exhaustive coverage in terms of potential toxicity pathways being affected, including xenobiotic and lipid metabolism and the thyroid hormone pathway. Herring gull eggs from Channel Shelter Island (CHSH, Lake Huron) and Gull Island (GULL, Lake Michigan) had among the highest OHC burdens, and extracts elicited a biochemical and transcriptomic response greater than that of extracts from the other three, less polluted colonies. For example, EROD EC50 values and porphyrin ECthreshold values were lower for CHSH and GULL extracts than for the other colonies. Extracts from CHSH and GULL altered 15 and 13 of 27 genes on the PCR array compared to no more than eight genes for the less contaminated sites. The combination of a well-established avian in vitro assay, two well-characterized biochemical assays, and the avian ToxChip PCR array permitted the geographical discrimination of variably contaminated herring gull eggs from the Great Lakes. Such high-throughput assays show potential promise as cost-effective tools for determining toxic potencies of complex mixtures in the environment.