A superhelical higher-order structure is proposed as a model of template DNA for regulation of transcription in simian virus 40. A stem-and-loop structure is located 3' downstream from the transcriptional initiation site in the sense strand. The complementary sequences to the loop regions in both strands are found between the 70th and the 300th nucleotides at the symmetrical sites as well as at the homologous sites with the stem-and-loop structure as the center. The complementary sequences are located such that both loops in sense and anti-sense strand encounter any of these sequences after one rotation of superhelix depending on the superhelical density. If each loop makes an intrastrand interaction with the complementary sequence at the symmetrical site either 5' upstream or 3' downstream, DNA forms a cruciform open-type structure at the root of the stem-and-loop region. When both loops interact with the complementary sequences at the homologous sites in both strands either 5' upstream or 3' downstream, a T-shaped closed-type structure is formed. The former is proposed as a signal for progression of transcription and the latter, cessation. If the transcription is defined as being regulated by the open- and the closed-structure at the stem-and-loop region which is constructed 3' downstream from the initiation site, the biochemical and genetic properties and activities of simian virus 40 genome are well interpreted.