Sumoylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. In Saccharomyces cerevisiae, but not in Schizosaccharyomes pombe, deletion of the gene encoding SUMO peptides is lethal. We have characterized the SUMO-encoding gene, sumO, in the filamentous fungus Aspergillus nidulans. The sumO gene was deleted in a diploid and sumOΔ haploids were recovered. The mutant was viable but exhibited impaired growth, reduced conidiation and self-sterility. Overexpression of epitope-tagged SumO peptides revealed multiple sumoylation targets in A. nidulans and SumO overexpression resulted in greatly increased levels of protein sumoylation without obvious phenotypic consequences. Using five-piece fusion PCR, we generated a gfp-sumO fusion gene expressed from the sumO promoter for live-cell imaging of GFP-SumO and GFP-SumO-conjugated proteins. Localization of GFP-SumO is dynamic, accumulating in punctate spots within the nucleus during interphase, lost at the onset of mitosis and re-accumulating during telophase. © 2007 Elsevier Inc. All rights reserved.