RNA polymerase is known to undergo a cycling process during initiation of transcription in vitro in which large amounts of small oligonucleotides are released. We have used this cycling reaction to determine the 5' end of the RNA synthesized from the outer ends of the Tn5 inverted repeats. By analyzing the size of the radiolabeled oligonucleotides synthesized using different labeled nucleoside triphosphates and in reactions deficient for a particular nucleoside triphosphate, the partial 5' sequence was obtained. This sequence was correlated with the DNA sequence of the region and an unambiguous origin for the mRNA was determined. The start site for the RNA, which is located at 97 base pairs from the outer ends of the inverted repeats, was confirmed by digesting gamma-32 P-ATP labeled full length (run off) transcripts with ribonuclease T1. The resulting gamma-labeled T1 generated oligonucleotide corresponded to the predicted size determined using the cycling reaction. With the knowledge of the RNA start site, the probable translation start sites for the protein(s) known to be required for Tn5 transposition can be predicted. Possible implications of the DNA sequence with respect to the regulation of the transposase are also discussed.