CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation. C ytosine methylation in CpG dinucleotides is an important mechanism of transcriptional regulation in vertebrates. Especially in the genome of mammalian somatic cells, the distribution of CpG dinucleotides, and the pattern of methylation are bimodal. In the major part of the genome, CpGs are underrepresented, dispersed, and predominantly methylated. Approximately 1-2% of the genome consists of nonmethylated CpG-rich stretches (CpG islands) that are typically 0.5-2 kb in length and usually associated with housekeeping genes (1, 2). This bimodal somatic methylation pattern is probably established by general de novo methylation, which skips CpG islands and adjacent gene promoters and leaves them unmethylated, and͞or by active demethylation of these sequences. Sp1 sites within the core sequence of CpG islands were shown to be required to prevent methylation (3, 4).Rous sarcoma virus (RSV) is an avian retrovirus that does not replicate in mammalian host cells. The nonpermissiveness of heterologous hosts is caused by several blocks of the replication cycle. Mammalian cells lack specific receptors for virus entry and do not promote correct splicing of retroviral mRNA, cleavage of viral polyproteins, or assembly of infectious virus particles on the inner surface of the cell membrane (reviewed in ref. 5). In addition to these obstacles, RSV proviral DNA is usually transcriptionally suppressed after integration into the mammalian genome. Fewer than 0.1% of RSV provirus-containing mammalian cells displays morphological transformation by virtue of the v-src oncogene (6, 7). The vast majority of RSV-infected cells harbors transcriptionally silent proviruses with undetectable amount of viral RNA (7, 8). Even in transformed mammalian cells, the proviral expression often tends to spontaneou...