The use of complementary RNA sequences such as antisense RNAs and ribozymes to regulate the expression of specific genes in eukaryotic cells has been well-documented, particularly with their application to both human gene therapy and plant biotechnology. Despite the simplicity of this approach, this technique usually results in only partial suppression of gene expression and, in some instances, even fails to regulate the gene of interest. The variation observed with antisense RNA and ribozyme-mediated regulation is further complicated by the many factors with the potential to impact on the effectiveness of these RNAs. Recent advances in the understanding of the global architecture of the nucleus, chromatin structure, and RNA metabolism provide useful and necessary information for designing novel approaches to improving antisense RNA and ribozyme regulation. These studies predict that the position of genes within the nucleus is not random and that transcripts produced from these genes follow specific tracks in migrating to the cell cytoplasm. These observations have the potential to impact significantly on the ways in which RNA-mediated forms of gene regulation are applied. The purpose of this review is to discuss the concept of colocalizing antisense RNAs and ribozymes with their target mRNAs and to introduce a variety of approaches aimed at achieving this goal.