Chemically synthesized oligonucleotides are powerful tools in the molecular biologist’s repertoire. This chapter describes the design of oligonucleotides and their applications, with particular reference to their use in the isolation and characterization of recombinant DNA clones. Examples of their use are taken either from the literature or from work carried out in our laboratory. Two specific areas are covered: the design of oligonucleotides, based on peptide sequence derived from a purified protein, for use in the isolation of clones from recombinant DNA libraries, and the design of consensus oligonucleotide probes, corresponding to conserved sequences in multigene families, for use in the isolation and characterization of such related genes. We do not cover the use of oligonucleotides for the localization of mRNA transcripts by in situ hybridization or for the analysis of mRNA steady-state levels by Northern blot analysis since this has been described elsewhere (1 ,2 ). A detailed discussion of all of the factors involved in the formation and melting of oligonucleotide/target DNA duplexes is beyond the scope of this treatise; for this, readers are referred to the work of Lathe (3 ). However, formulae for the calculation of melting temperatures and the application of the base analog inosine are described in the Notes.