The state of viral DNA in three Marek's disease virus (MDV)-transformed chicken cell lines (MDCC-MSB-1, MDCC-HP-2, MDCC-RP-1) was investigated by CsCl-density gradient centrifugation, and sedimentation analysis in neutral glycerol gradients. Each cell line contained MDV DNA integrated into the host cell genome. Additionally, free viral DNA could be identified in MDCC-MSB-1 and MDCC-HP-2 cells, sedimenting at about 100 S, and banding at the position of circular DNA in CsCl-ethidiumbromide gradients. Thus, MDV DNA with properties of circular plasmid DNA could be demonstrated in 2 virus-transformed cell lines. The significance of circular plasmid forms of viral DNA remains yet to be clarified, since these are apparently not regularly found in MDV-transformed cells.