Abstract Recently it was discovered that the perfluorooctane sulfonate (PFOS) detected in wildlife, such as fish-eating birds, had a greater proportion of linear PFOS (L-PFOS) than the manufactured technical product (T-PFOS), which contains linear and branched isomers. This suggests toxicological studies based on T-PFOS data may inaccurately assess exposure risk to wildlife. To determine whether PFOS effects were influenced by isomer content, we compared the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either L-PFOS or T-PFOS using Agilent microarrays. At equal concentrations (10 µM), T-PFOS altered the expression of more transcripts (340, >1.5-fold change, p < 0.05) compared with L-PFOS (130 transcripts). Higher concentrations of L-PFOS (40 µM) were also less transcriptionally disruptive (217 transcripts) than T-PFOS at 10 µM. Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, hepatic system development, and cellular growth and proliferation. Pathway and interactome analysis suggested that genes may be affected through the RXR receptor, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling, and PPARγ and SREBP receptors. In all functional categories and pathways examined, the response elicited by T-PFOS was greater than that of L-PFOS. These data show that T-PFOS elicits a greater transcriptional response in CEH than L-PFOS alone and demonstrates the importance of considering the isomer-specific toxicological properties of PFOS when assessing exposure risk. Environ. Toxicol. Chem. 2011;30:2846–2859. © 2011 SETAC