The attachment of the ubiquitin-like protein SUMO to target proteins is involved in a number of important cellular processes. Typically, SUMO modification occurs on lysine residues within the consensus sequence psiKxE/D (psi is a hydrophobic residue and x is any residue), although there are examples of modifications at nonconsensus sites. In most cases, sites of SUMO modification have been inferred from a combination of site-directed mutagenesis and functional analysis; however, these methods have two limitations. They do not directly identify the acceptor lysine, nor are they sufficient to identify acceptor lysine residues in SUMO polymers. Here, we use Fourier transform ion cyclotron resonance (FT-ICR) together with activated-ion electron capture dissociation (AI-ECD) or infrared multiphoton dissociation (IRMPD) mass spectrometry techniques to overcome these restrictions. These approaches were employed to analyze the autoSUMOylation reaction catalyzed by the SUMO E3 ligase RanBP2. Six sites of in vitro SUMOylation in RanBP2 along with four branch-point lysines in SUMO-1 and three in SUMO-2 were identified. In all but one case, SUMOylation occurred within the sequences KxE or KpsiK. These results demonstrate the utility of FT-ICR with AI-ECD or IRMPD mass spectrometry in detecting SUMOylation, and sites of SUMOylation, and their potential roles as complementary tools for proteomic and functional analysis, and provide significant insight into the modification of a SUMO ligase for which conventional techniques have been unsuccessful.