Publisher Summary This chapter presents the tetracycline-regulatable promoters in yeast, and describes its use for functional analysis using the Sit4 and Ppz1 Ser/Thr protein phosphatases as an example. The chapter discusses the regulation of gene expression in yeast cells, using the Tet system. The Tet system is being used for the regulation of gene expression in cells of different organisms (such as yeasts, plants, Drosophila, or mammals), under the control of doxycycline and other antibiotic molecules of the tetracycline family. In its initial form, it was designed for regulated expression in mammalian cells. It is based on the activity of a hybrid transcriptional regulator (tTA) formed by the TetR DNA-binding domain (the tetracycline-inducible repressor from the Escherichia coli Tn10 transposon) fused to the VP16 activator domain of the herpes simplex virus. Construction and phenotypic analysis of a sit4 hal3 conditional mutant by using a one-step promoter substitution cassette and the Tet-regulatable dual expression system is also discussed in the chapter.