Analysis of the function of a particular gene product typically involves determining the expression profile of the gene, the subcellular location of the protein, and the phenotype of a null strain lacking the protein. Conditional alleles of the gene are often created as an additional tool. We have developed a multifunctional, transposon-based system that simultaneously generates constructs for all the above analyses and is suitable for mutagenesis of any given Saccharomyces cerevisiae gene. Depending on the transposon used, the yeast gene is fused to a coding region for beta-galactosidase or green fluorescent protein. Gene expression can therefore be monitored by chemical or fluorescence assays. The transposons create insertion mutations in the target gene, allowing phenotypic analysis. The transposon can be reduced by cre-lox site-specific recombination to a smaller element that leaves an epitope tag inserted in the encoded protein. In addition to its utility for a variety of immunodetection purposes, the epitope tag element also has the potential to create conditional alleles of the target gene. We demonstrate these features of the transposons by mutagenesis of the SPA2, ARP100, SER1, and BDF1 genes.