Promoter locations for the T3 RNA polymerase on the physical map of T3 DNa have been determined. Through the use of conditions favoring the synthesis of RNA from the class II region, an agarose-formaldehyde gel system which improves the resolution of high molecular weight RNAs, and template DNA that was cut by one of several restriction endonucleases prior to transcription, seventeen promoter locations for the T3 RNA polymerase have been mapped. Ten promoters have been identified in the class II region and one promotor has been identified in the class II region and one promotor has been identified in the early (class I) region. The locations of previously mapped class III promoters and the internal termination signal for the T3 RNA polymerase have been mapped more precisely than in previous reports. The resulting transcription map demonstrates a striking similarity to the transcription map of bacteriophage T7.