To investigate the regulation of Escherichia coli topA gene transcription, primer extension was employed to determine the transcription initiation sites from the chromosomal topA gene. When cells were grown in LB medium to log phase, four transcription initiation sites could be identified. Three of these sites corresponded to promoters P1, P2 and P4 previously characterized using topA-galK fusion plasmids. The P3 promoter that is active on the plasmid was not utilized at the chromosomal topA gene under the conditions employed. There was a new transcription initiation site corresponding to a new promoter Px1. When cells started to enter stationary phase, promoter Px1 gradually became the major transcription initiation site for topA, while transcription from promoters P2 and P4 decreased. In an E. coli mutant lacking sigmaS (the rpoS gene product), the stationary phase specific sigma factor, the induction of transcription from promoter Px1 was abolished. In another mutant lacking H-NS activity, resulting in increased sigmaS level in log-phase, the transcription from promoter Px1 during log phase was increased. Thus Px1 appeared to be regulated by sigmaS. The activity of promoter P1 on the chromosome increased during heat shock, consistent with the previous result obtained using the topA-galK fusion plasmid showing that P1 is a sigma32-dependent heat shock promoter. Promoters P2 and P4 were most likely to be recognized by sigma70. The total level of topoisomerase I protein in the rpoS mutant was not reduced significantly in stationary phase due to increased transcription initiation from the other topA promoters. The utilization of multiple sigma factors for transcription initiation of topA could be important for adaptation of E. coli to change in growth conditions.