The major transactivator protein IE2p86 of human cytomegalovirus (HCMV) has previously been shown to undergo posttranslational modification by the covalent attachment of SUMO proteins, termed SUMOylation, which occurs at two lysine residues located at amino acid positions 175 and 180. Mutation of the acceptor lysines resulted in the abrogation of IE2p86 SUMOylation in mammalian cells and a strong reduction of IE2p86-mediated transactivation. In this paper, we identify an additional SUMO interaction motif (SIM) within IE2p86, which mediates noncovalent binding to SUMO, as shown by yeast two-hybrid analyses. Transient-expression experiments revealed that an IE2p86 SIM mutant exhibited significantly reduced SUMOylation, strongly suggesting that noncovalent SUMO interactions affect the efficacy of covalent SUMO coupling. In order to define the relevance of IE2p86 SUMO interactions for viral replication, recombinant viruses originating from two different HCMV strains (AD169 and VR1814) were generated. Analysis of viruses expressing SUMOylation-negative IE2p86 revealed strongly impaired replication due to reduced viral DNA and protein accumulation, as well as diminished initiation of immediate-early gene expression. The additional introduction of the SIM mutation into the viral genome did not further compromise viral replication but resulted in altered expression of viral proteins at late times postinfection. In summary, this paper clearly shows that IE2p86 SUMOylation is necessary for efficient replication of the HCMV laboratory strain AD169 and the clinical isolate VR1814 and thus for the in vivo function of this viral transcription factor.