The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which organochlorine contaminants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and some polycyclic aromatic hydrocarbons induce toxicity and altered gene expression. Atlantic salmon has multiple AHR genes, of which two belong to the AHR1 clade and four belong to the AHR2 clade. The four AHR2 forms (alpha, beta, gamma, delta) are more highly expressed than the AHR1 (alpha, beta,) forms and all six AHRs are highly similar in pairs, likely originating from a whole-genome duplication in the salmonid ancestor. It has been speculated that having multiple AHRs contributes to the very high sensitivity of salmonid species to TCDD and related chemicals. To test the hypothesis that all four salmon AHR2 proteins are expressed and functional, we measured mRNA transcription for each AHR2 in several tissues, cloned the cDNAs and evaluated the functional properties of the expressed proteins. Analysis by real-time PCR revealed that the receptors showed differences in transcript levels among salmon tissues and that in general AHR2alpha was transcribed at higher levels than the other three AHR2s. Velocity sedimentation analysis showed that all four in vitro-expressed AHR2 proteins exhibit specific, high-affinity binding of [(3)H]TCDD. When expressed in COS-7 cells, all four AHR2 proteins were able to drive the expression of a reporter gene under control of murine CYP1A1 enhancer elements. From EC(50) values determined in TCDD concentration-response experiments, all four salmon AHR2s show similar sensitivity to TCDD. In summary, all four Atlantic salmon AHR2 appear to function in AHR-mediated signaling, suggesting that all four proteins are involved in TCDD-mediated toxicity.