Transcriptional initiation sites of the ostA gene involved in organic solvent sensitivity in Escherichia coli were found by primer extension analysis. Two transcriptional initiation sites were newly identified at -133 and -48 nucleotides from the initiation codon of ostA, but the previously reported sigma(E)-dependent one at -227 could not be detected. No heat-inducible expression of ostA was observed by Northern blotting analysis, indicating that the contribution of sigma(E)-dependent transcription was very small if any. sigma(D)-Dependent promoter-like sequences were found just upstream of the newly identified transcriptional initiation sites by computer-aided analysis. Deletion analysis of ostA-lacZ fusions demonstrated that these two promoters contributed almost equally to the constitutive expression of the ostA gene.