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This chapter describes a transposon mutagenesis system that produces multipurpose constructs for the monitoring of protein production, localization, and function. A single mutagenesis generates a large spectrum of alleles, including null, hypomorphic, and conditional alleles, reporter fusions, and epitope-insertion alleles. The system, therefore, provides the basis for a wide variety of studies of gene and protein function. The chapter provides comprehensive instructions for use of the new transposons to mutagenize a gene of interest, and for use of the transposon insertion libraries to mutagenize the yeast genome. While the application of these specific transposons is limited to organisms in which the Saccharomyces cerevisiae selectable marker URA3 can be used, the approach is generally applicable to mutagenesis of DNA from any organism for which a transformation and selection system exists.