To study the autonomous replication of constructs combining replication and transcription elements, hybrid plasmids were designed to contain both higher eukaryotic ori/ARS and the cytomegalovirus (CMV) promoter-enhancer. In pCI.ARS-neo, ARS was from autonomously replicating stable transgene pr8a of silkworm Bombyx mori. In pCI.ori-neo, chromosomal ori was from the human c-myc locus. Analysis of the maintenance of pCI.ARS-neo and pCI.ori-neo in transgenic mice and in cultured immortalized human T cells revealed the interplay of transcription and replication, as the CMV element suppressed autonomous replication of both plasmids. This might reflect the conflict between transcription and replication of the genome in higher eukaryotic cells.