DNA from bacteriophage SP6 grown on Salmonella typhimurium LT2 is a linear duplex with a unique DNA sequence having a molecular weight of 2.9 x 10(6) (43.500 base pairs). Restriction endonuclease cleavage maps on SP6 DNA for Ava II, Kpn I, Bgl II, Eco RI, and HindIII have been determined. SP6 DNA is transcribed selectively in vitro by Escherichia coli RNA polymerase, predominantly from three strong promoter sites located near the left end of the standard physical map, reading rightward to a termination site near 6,000 base pairs. Transcription in vitro by purified SP6-specific RNA polymerase gives rise to at least 10 discrete RNA species, all of which are read rightward. Promoter sites for these species are located throughout the rightmost 90% of the SP6 DNA molecule, although precise mapping has not yet been carried out. The general form of the SP6 transcriptional map is similar to the T7- and T3-like phages, although no gross sequence homologies are evident from DNA-RNA hybridization experiments.