Ribosome display is a cell-free technology for the in vitro selection and evolution of proteins encoded by DNA libraries, in which individual nascent proteins (phenotypes) are linked physically to their corresponding mRNA (genotypes) in stable protein-ribosome-mRNA (PRM) complexes. Formation of the complexes can be achieved through deletion of the stop codon of the mRNA, stalling the ribosome at the end of translation; the nascent protein is extended by a spacer such as the immunoglobulin Ckappa domain or others to allow exit through the ribosome tunnel. Through affinity for a ligand, the protein-mRNA coupling permits simultaneous isolation of a functional nascent protein and its translated mRNA; the latter is then converted into cDNA by reverse transcription and amplified for further manipulation, repeated cycles or soluble protein expression. Through the use of PCR-generated libraries, avoiding the need for cloning, ribosome display can be used to both screen very large populations and continuously search for new diversity during subsequent rounds of selection. Additionally, the use of cell-free systems allows the selection of proteins that are toxic or unstable in cells, and proteins with chemical modifications. Ribosome display systems using both prokaryotic and eukaryotic cell extracts have been developed. Examples of the application of eukaryotic systems include the selection and evolution of antibody fragments, DNA binding domains, enzymes, interacting proteins and peptides among others. Here we describe the step-by-step procedure to perform our previously described eukaryotic ribosome display method, which has the distinctive feature of an in situ reverse transcription-PCR (RT-PCR) procedure for DNA recovery from ribosome-bound mRNA. We also introduce a recent, previously unpublished improvement to the procedure in which in situ reverse transcription is combined with sensitive single-primer PCR technology.