PrPC, the cellular isoform of prion protein, is widely expressed in most tissues, including brain, muscle and gastrointestinal tract. Despite its involvement in several bioprocesses, PrP has still no apparent physiological role. During propagation of transmissible spongiform encephalopathies (TSE), prion protein is converted to the pathological isoform, PrPSc, in a process believed to be mediated by unknown host factors. The identification of proteins associated with PrP may provide information about both the biology of prions and the pathogenesis of TSE. Thus far, PrPC has been shown to interact with synaptic proteins, components of the cytoskeleton and intracellular proteins involved in signalling pathways. Here, we describe the association of PrP with the β4 subunit of nicotinic acetylcholine receptor (nAChR), as indicated by co-immunoprecipitation assays and double-label immunofluorescence. The interaction between prion protein and native β4 subunit was further studied by affinity chromatography, using immobilized and refolded recombinant PrP as a bait and brain homogenates from normal individuals. Additionally, the participation of β4 subunit in the pathogenesis of TSE was studied by in vivo assays. β4–/– and wild-type mice were challenged with the RML (Rocky Mountain Laboratories) infectious agent. Transgenic animals displayed altered incubation times but the deletion of β4 subunit did not result in a significant change of the incubation period of the disease. Our results suggest that PrPC is a member of a multiprotein membrane complex participating in the formation and function of α3β4 nAChR.