Prokaryotic and eukaryotic in vitro translation systems have recently become the focus of increasing interest for tackling fundamental problems in biochemistry. Cell-free systems can now be used to study the in vitro assembly of membrane proteins and viral particles, rapidly produce and analyze protein mutants, and enlarge the genetic code by incorporating unnatural amino acids. Using in vitro translation systems, display techniques of great potential have been developed for protein selection and evolution. Furthermore, progress has been made to efficiently produce proteins in batch or continuous cell-free translation systems and to elucidate the molecular causes of low yield and find possible solutions for this problem.