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The use of transposons allows the rapid construction of a large number of the alleles of a gene of interest. Transposon insertion libraries can be used for both the mutagenesis and identification of genes regulated by particular growth conditions and strain backgrounds. This chapter presents an overview of a newly developed multipurpose transposon mutagenesis system that allows the monitoring of protein production, function, and localization in yeast. The system uses two basic types of transposon, designated as mTn-3×HA/lacZ and mTn 3×HA/GFP. The transposon system was tested by the mutagenesis of several individual yeast genes. The HAT tag was successfully used to analyze the localization of the Spa2, Arp100, and Sao1 proteins. The new transposons expand the repertoire of insertions that may be generated to include GFT fusions and epitope tags. It is expected that shuttle …