HEPES is a well-known buffering reagent used in cell-culture medium. Interestingly, this compound is also responsible for significant modifications of biological parameters such as uptake of organic molecules, alteration of oxidative stress mechanisms or inhibition of ion channels. While using cell-culture medium supplemented with HEPES on prion-infected cells, it was noticed that there was a significant concentration-dependent inhibition of accumulation of the abnormal isoform of the prion protein (PrP(Sc)). This effect was present only in live cells and was thought to be related to modification of the PrP environment or biology. These results could modify the interpretation of cell-culture assays of prion therapeutic agents, as well as of previous cell biology results obtained in the field using HEPES buffers. This inhibitory effect of HEPES could also be exploited to prevent contamination or propagation of prions in cell culture.