Prion and other amyloid-forming diseases represent a group of neurodegenerative disorders that affect both animals and humans. The role of metal ions, especially copper and zinc is studied intensively in connection with these diseases. Their involvement in protein misfolding and aggregation and their role in creation of reactive oxygen species have been shown. Recent data also show that metal ions not only bind the proteins with high affinity, but also modify their biochemical properties, making them important players in prion-related diseases. In particular, the level of zinc ions is tightly regulated by several mechanisms, including transporter proteins and low molecular mass thiol-rich proteins, which in brain tissue is called metallothioneins. From four metallothionein isoforms, metallothionein-3, a unique brain-specific metalloprotein, plays a crucial role only in this regulation. This review critically evaluates the involvement of metallothioneins in prion- and amyloid-related diseases in connection with metal ions and regulation of their homeostasis. Moreover, the relationship between isoforms is discussed as well as in the perspective of their simultaneous analysis.