Transposons are mobile genetic elements with the capacity to “jump” to new target DNA. Although first discovered in Zea mays by McClintock (1 ), they are present in DNA genomes of species from all kingdoms. Transposons fall into two major classes. Class I transposons are retroelements that transpose via an RNA intermediate that is synthesized by a reverse transcriptase (e.g., Alu elements in primates and Ty elements in yeast); class II transposons transpose directly from DNA to DNA (e.g., P elements in yeast and Tn elements in bacteria). This chapter describes the application of class II Tn elements from bacteria to mutagenize plasmid DNA by insertional mutagenesis.