We describe the quantitative monitoring of TATA-binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) x TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM). When GFP x TBP expression was altered by using various promoters, the levels measured by LSCM correlated well with the levels determined by immunoblot of whole cell extract protein. These results show that GFP x TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real-time quantitation in living cells and subcellular localization information. Time-lapse confocal imaging of GFP x TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0.7) between mother and daughter cells. Based on this and data from a mutant which underexpresses GFP x TBP, we suggest that intracellular levels of TBP are near rate-limiting for growth and viability.