Episomal plasmids for stable transfection of mammalian cell cultures were constructed that have a G418-resistance (neo) gene immediately downstream of a highly truncated promoter. These plasmids had a function hygromycin-resistance gene (hyg) as a selectable marker. Surprisingly, in LTK- cells, but not HeLa cells, stably transfected with these BK virus-based plasmids having no promoter elements adjacent to the neo gene, readthrough transcription, probably from about 1 kb upstream, gave almost as efficient expression of the neo gene as of the hyg gene with a full-length promoter immediately upstream. When the transfecting plasmids contained Epstein-Barr virus (EBV) DNA sequences for episomal maintenance and had multiple Sp1 sites and a TATA box as the only promoter elements 5' to the neo gene, only about 3-9% of HeLa transfectants were G418 resistant (G418R). In transfections with analogous plasmids lacking these promoter elements 5' to the neo gene, no G418R colonies were seen. The establishment of the G418R phenotype probably required integration of plasmid DNA into favorable chromosomal sites and was aided by the presence of the TATA box plus Sp1 sites as a subminimal promoter. The absence of detectable G418-resistance in most of the HeLa transfectant clones obtained with EBV-type plasmids, even at a high plasmid copy number and even when a TATA box and six Sp1 sites were present immediately upstream of the neo gene, indicates that these elements do not suffice for appreciable gene expression in vivo and that this is a suitable model system for studying DNA rearrangements that can potentiate expression of the neo gene.