We previously described the integration of a nonacute retrovirus, reticuloendotheliosis virus (REV), into the genome of a herpesvirus, Marek disease virus (MDV), following both long-term and short-term coinfection in cultured fibroblasts. The long-term coinfection occurred in the course of attenuating the oncogenicity of the JM strain of MDV and was sustained for > 100 passages. The short-term coinfection, which spanned only 16 passages, was designed to recreate the insertion phenomenon under controlled conditions. We found that REV integrations into MDV were common and could occur within the first passage following coinfection. Now we have mapped the integration sites. After 5-16 passages in vitro, 17 out of 19 REV junction sites are clustered in two 1-kilobase regions at the junctions of the short unique and short repeat region of MDV. In the long-term cocultivation experiment, 6 out of 10 insertions also mapped in this region. In both cases, integrated proviruses are unstable and undergo subsequent recombinative deletion, often leaving a solitary long terminal repeat. The long terminal repeat sequences are, however, stably maintained for many rounds of passaging in vitro. This clustering of insertions presumably is influenced by selection for viable and fast-growing viruses, and occurs in a region of the MDV genome which shows significant size heterogeneity in several strains.