50 kb of contiguous DNA sequences covering the human c-myc coding region and approximately 20 kb of flanking upstream and downstream sequences were cloned onto a prokaryotic F-factor derived plasmid, which also contains a selectable marker and the plasmid origin of DNA replication oriP of Epstein Barr virus (EBV). Since these plasmids replicate extrachromosomally after stable transfection into EBV-positive B-cell lines, the gene regulation of c-myc can be analysed independent from chromosomal integration positions. Despite the presence of all known c-myc regulatory elements on these constructs, expression from the stably transfected c-myc gene was barely detectable in either cell line. Hypermethylation of these plasmids could be excluded as a mechanism for the lack of gene expression. Insertion of the immunoglobulin kappa-intron and 3' enhancers, however, activated c-myc transcription, when placed adjacent to or separated from the c-myc promoters by as far as 30 kb. These results indicate that transcription of c-myc in vivo requires additional and still unidentified control elements located outside this 50 kb fragment, and experimentally demonstrate long range enhancer function in vivo.