The Escherichia coli gene dnaQ coding for the epsilon subunit of DNA polymerase III was suppressed in vivo via antisense RNAs. To this aim, different fragments of dnaQ were cloned in reverse orientation into the pBAD-HisB vector or into pT3T7, and the corresponding antisense RNAs were conditionally expressed in vivo. Antisense transcripts featuring fast hybridization in vitro with dnaQ mRNA but lacking stability in vivo did not suppress the target gene. Moreover, the in vivo concentration of an antisense transcript was positively correlated to its silencing effectiveness.