Early identification of toxicity associated with new chemical entities is important for reducing compound attrition in late stage drug discovery. Activation of the aryl hydrocarbon receptor (AhR) by xenobiotics is a recognised mechanism of toxicity: the AhR mediates most, if not all, of the serious toxicities caused by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition to compounds such as TCDD, the AhR can be activated by compounds with drug-like properties; consequently there is a desire to eliminate AhR activity in candidate drug programs. Endogenous AhR translocates from the cytoplasm to the nucleus in response to prototypical AhR ligands. This trafficking was monitored in mouse Hepa-1 cells, human HepG2 cells and rat primary hepatocytes using an anti-AhR antibody. A confocal imaging plate reader, the InCell Analyzer 3000, was used to image fixed cells cultured in 96 well plates, and algorithms were used to analyse both population data and individual cell responses. The subsequent induction of the CYP1A1 gene, in the three cell models, was also assessed using quantitative real-time polymerase chain reaction and showed good correlation with the translocation assay. To conclude, we have established robust, automated high throughput assays for the identification of AhR activators in primary hepatocytes and cell lines.