We have examined transcriptional start sites responsible for expression of the transposase and transposition inhibitor proteins encoded by IS50R, and determined the likely translational start site of transposase. Amino-terminal analysis of a transposase-beta-galactosidase fusion protein gave the sequence Met-Ile-Thr-Ser-Ala, which corresponds to the predicted amino acid sequence starting at position 93 of IS50. S1 nuclease mapping of IS50 RNA produced in vivo indicated that three transcripts, T1, T2 and T3, start near this position. Only T1 starts upstream from the transposase amino terminus. T2 corresponds to an in-vitro transcript described previously. Analysis of the transcripts and proteins produced from deletion derivatives of an IS50-lacZ construct suggested that the three transcripts initiate at independent but overlapping promoters clustered near the end of IS50. This analysis confirmed that only T1 can encode transposase, and that T2 is largely responsible for expression of the inhibitor protein. The coding capacity of T3 was not determined. Finally, transcripts that originate outside of IS50 are prevented from expressing transposase because of a secondary structure that is present in these transcripts only.