We have investigated the copy number, chromosomal subunit conformation and regulation of expression of integrated avian retrovirus genomes. Our results indicate that there are approximately two copies of the endogenous viral genomes (RAV-O) per haploid cell genome in uninfected chick embryo fibroblasts (CEF) and red blood cells (RBC). The copy number and subunit conformation (as measured by DNAasel sensitivity) of the RAV-O genomes are independent of the level of expression of these viral DNA sequences. In cells isolated from embryos of the V+, gs-chf- and gs+chf+ phenotypes, approximately one of the two viral genomes is in a DNAase l-sensitive conformation. Upon infection with an exogenous Rous sarcoma virus (PR-RSV-C), one new viral genome is integrated per haploid CEF genome. The newly integrated RSV genome is completely sensitive to DNAase l, and the subunit conformation of the endogenous viral genomes is not altered by the integration of additional exogenous proviruses. Both the endogenous and newly integrated exogenous viral genomes are present in "nu-body" structures, and the selective sensitivity of these proviral DNA sequences to DNAase l is maintained in isolated nucleosomes. Our experiments revealing the DNAase l sensitivity of one of the two RAV-O genomes in gs-chf-CEF led us to reexamine the level of viral specific RNA in CEF of various GS genotypes. We find that GS/GS CEF contain approximately 100 copies of viral RNA per cell, gs/gs CEF contain no detectable viral RNA, and the heterozygote GS/gs CEF contain approximately 50 copies of viral specific RNA per cell. These results suggest that the GS gene controls production of RAV-O RNA sequences in CEF in a "cis" fashion. In RBCs, however, the expression of the RAV-O genome is independent of the GS gene, with both GS/GS and gs/gs RBCs containing roughly equivalent amounts of viral specific RNA. Our results suggest that the chromosomal structure of the endogenous viral genes is independent of the GS gene, and that the GS gene is cis-acting and tissue-specific.