Mapping analysis of the nucleosomal organization of integrated human papillomavirus type 18 (HPV18) DNA in HeLa cells reveals a very prominent nuclease-hypersensitive site within the viral noncoding regulatory region that harbors transcriptional control sequences and coincides with most of the 5' ends of the cytoplasmic early mRNAs. Moreover, it is shown that the conserved coamplified 5' cellular flank, common to all HPV18 copies in HeLa cells and located close to the virus-cell integration site, also contains several distinct hypersensitive sites, accessible not only to DNase I but also to restriction enzymes. Nuclear run-on analysis in isolated HeLa nuclei demonstrates the occurrence of nascent transcripts covering the cellular flank (the late and the viral noncoding regulatory region), indicating that a cellular promoter, marked by the hypersensitive sites, cooperates with the viral control region in generating the HPV18 transcripts. Cycloheximide treatment of HeLa cells results in a reduction of the cytoplasmic steady-state level of the 3.5-kb mRNA corresponding to the viral E6, E7, and parts of the E1 open reading frames (ORFs), whereas the expression of the 1.6-kb transcript corresponding only to the E6 and E7 ORFs is not influenced. Nuclear run-on analysis carried out after the cycloheximide chase reveals that the distribution of nascent transcripts spanning the viral E6, E7, and parts of the E1 region is substantially decreased. In contrast to this finding, an even, pronounced increase of the elongation rate of those transcripts, which cover the cellular flank, the late and the viral noncoding regulatory region was noted indicating a different involvement of regulatory factors in the activity of both promoters.