The functional significance of changes in nuclear structure and organization in transformed cells remains one of the most enigmatic questions in cancer biology. In this review, we discuss relationships between nuclear organization and transcription in terms of the three‐dimensional arrangement of genes in the interphase cancer nucleus and the regulatory functions of nuclear matrix proteins. We also analyse the role of nuclear topology in the generation of gene fusions. We speculate that this type of multi‐layered analysis will one day provide a framework for a more comprehensive understanding of the genetic origins of cancer and the identification of new therapeutic targets. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.