Epithelial cells are important in inflammation and immunity. In this study, we examined if Madin–Darby canine kidney (MDCK) epithelial cells express functional P2X7 receptors, which bind the damage-associated molecular pattern extracellular adenosine 5′-triphosphate (ATP). Reverse transcription (RT)-PCR and immunoblotting revealed the expression of P2X7 in MDCK cells. A flow cytometric assay demonstrated that ATP or 2′(3′)-O-(4-benzoylbenzoyl)ATP induced ethidium+ uptake into MDCK cells, and that this process was impaired by the P2X7 antagonists KN-62 and A438079. RT-PCR also demonstrated the presence of Toll-like receptor 4, NALP3, caspase-1, interleukin-1β and interleukin-18 in MDCK cells, as well as in positive control LPS-primed canine monocytes. In conclusion, the MDCK epithelial cell line expresses functional P2X7, as well as Toll-like receptor 4 and molecules associated with the NALP3 inflammasome. This cell line may help elucidate the role of these molecules in kidney epithelial cells and renal disorders in dogs and humans.