ColE1-like plasmids are widely used as expression vectors and as gene delivery vehicles. We have recently described a naturally occurring plasmid deletion phenomenon in the ColE1-type plasmid, pCI-neo, which leads to the detectable expression of an apparently promotorless kanamycin resistance gene. In the current work, we found that the expression of that aminoglycoside phosphotransferase (aph) gene is regulated by an RNAII preprimer promoter located within the plasmid ColE1 replication origin, as a consequence of the extension of the RNA II species for at least 1.5 kb, up to the aph gene. This mechanism is dependent on the nonformation and/or dissociation of the hybrid between plasmid DNA and RNA II preprimer transcript. This is the first in vivo description of RNA II transcription beyond ori in wild-type Escherichia coli strains and nonmutated RNAII plasmid sequences resulting in productive transcription of distant downstream genes. Our results raise questions about unwanted expression of genes from expression or cloning vectors of ColE1 type and highlight the importance of a more careful design of ColE1-derived plasmid vectors.