SUMO, or Smt3 in Saccharomyces cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo. Many of these substrate modifications are cell cycle-regulated, and SUMO conjugation is essential for viability in most eukaryotes. However, only a limited number of SUMO-modified proteins have been definitively identified to date, and this has hampered study of the mechanisms by which SUMO ligation regulates specific cellular pathways. Here we use a combination of yeast two-hybrid screening, a high copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast. These three approaches yielded overlapping sets of proteins with the most extensive set by far being those identified by mass spectrometry. The two-hybrid data also yielded a potential SUMO-binding motif. Functional categories of SUMO-modified proteins include SUMO conjugation system enzymes, chromatin- and gene silencing-related factors, DNA repair and genome stability proteins, stress-related proteins, transcription factors, proteins involved in translation and RNA metabolism, and a variety of metabolic enzymes. The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms.