Two functional promoters had previously been identified in the mobile genetic element IS30 of Escherichia coli. One, P30A, controls the transcription of ORF-A, whose product is the transposase; the other, located within the ORF-A sequence but on the opposite strand, is called P30C, but the nature and function of its product had remained unknown. We identified this product as an RNA about 150 nucleotides long (called RNA-C) that functions as an untranslated antisense transcript. Indeed, biochemical evidence indicates that ORF-C, which is completely contained on RNA-C, is not translated at detectable levels. Mutational analysis of P30C revealed that overproduction of RNA-C resulted in a decrease of IS30 transposition, while a reduction in the promoter strength resulted in an increase of transposition, as measured by the rate of cointegrate formation. We showed that the translation of ORF-A, but not transcription, is negatively affected by the presence of antisense RNA-C. In contrast to other antisense RNAs acting inhibitorily on translation, RNA-C does not seem to affect translation initiation. Most likely its hybridization to the transposase mRNA in the complementary region located in the central part of ORF-A inhibits the ribosomes in their progression, thus reducing the number of completely translated transposase molecules.