Transcription was carried out in vitro by RNA polymerase on supercoiled minichromosome DNA containing the replication origin of the Escherichia coli chromosome (oriC) and the products were analyzed. Leftward transcription starting from the 16-kDa gene promoter located adjacent to the right of oriC was inhibited by the DnaA protein, a protein essential for initiation of replication. This inhibition is due to binding of DnaA protein to the 9-base pair sequence (dnaA box) located just upstream of the 16-kDa gene promoter. The inhibition was observed at levels of DnaA protein and RNA polymerase comparable to those required for replication of oriC plasmids in vitro and was abolished at high levels of RNA polymerase. These results suggest that a balance of DnaA protein and RNA polymerase is important in regulating transcription and that this regulation operates under conditions that allow initiation of replication. The transcription traversed oriC with very few transcripts terminating before or within it. DnaA protein did not affect the chain length of these transcripts. Furthermore, we identified leftward transcripts which start close to position 177 in the oriC sequence.