The partially matched I and O ends of IS50 (the insertion sequence of the transposon Tn5) are needed for transposition, probably as the sites upon which the cis-acting transposase and host proteins act. To better understand how transposition is regulated we made a series of IS50-related elements in which the positions of the ends and of the transposase gene were varied systematically. Assays of these elements showed that the I and O ends differ inherently in transposition activity. Other workers showed that methylation, at DNA N6-adenine methyltransferase (Dam) recognition sites within the I end and the transposase tnp gene promoter, inhibits transposase synthesis and also I end activity. We show that the effect of Dammediated methylation on an I end depends on the end's orientation relative to the tnp gene. Further, in dam+ cells oriented like -tnp----in relation to the first and second ends) are (O, I) greater than (O, O) greater than or equal to (I, O) greater than (I, I). In dam- cells the relative activities are (O, I) = (I, O) = (I, I) greater than (O, O). Our results are consistent with a model orginally developed for IS10, in which hemi-methylation resulting from passage of a replication fork regulates transposition.