A relationship exists between nuclear architecture and gene activity and it has been proposed that the activity of ongoing RNA polymerase II transcription determines genome organization in the mammalian cell nucleus. Recently developed 3C and 4C technology allowed us to test the importance of transcription for nuclear architecture. We demonstrate that upon transcription inhibition binding of RNA polymerase II to gene regulatory elements is severely reduced. However, contracts between regulatory DNA elements and genes in the β-globin locus are unaffected and the locus still interacts with the same genomic regions elsewhere on the chromosome. This is a general phenomenon since the great majority of intra- and interchromosomal interactions with the ubiquitously expressed Rad23a gene are alos not affected. Our data demonstrate that without transcription the organization and modification of nucleosomes at active loci and the local binding of specific trans-acting factors is unaltered. We propose that these parameters, more than transcription of RNA polymerase II binding determine the maintenance of long-range DNA interactions.