The Wilms' tumour suppressor gene WT1 encodes multiple isoforms of a transcription factor essential for correct mammalian urogenital development. Maintenance of the correct isoform ratio is critical. In humans, perturbation of this ratio causes Frasier syndrome, which is characterized by developmental defects of the kidney and urogenital tract. Different WT1 isoforms are thought to regulate transcription and participate in mRNA processing, functions reflected by a complex sub-nuclear distribution. However, the role of individual WT1 isoforms remains unclear and pathways leading to WT1 sub-nuclear localization are completely unknown. Here we use cells expressing green fluorescent protein-tagged WT1 to demonstrate that the two major WT1 isoforms occupy separate and dynamic intranuclear locations in which one isoform, WT1+KTS, preferentially associates with the nucleolus. The alternatively spliced zinc finger region is found to be critical for the initial sub-nuclear separation of WT1 isoforms, but interactions between different isoforms influence the sub-nuclear distribution of WT1. We illustrate how disruption of WT1 nuclear distribution might result in disease. This study contributes to the emerging picture of intranuclear protein trafficking.