The spx gene of Bacillus subtilis encodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It is located in a dicistronic operon with the yjbC gene. The spx gene DNA complements an spx null mutation with respect to disulfide stress resistance, suggesting that spx is transcribed from a promoter located in the intergenic region of yjbC and spx. Transcription of the yjbC-spx operon has been reported to be driven by four promoters, three (P(1), P(2), and P(B)) residing upstream of yjbC and one (P(M)) located in the intergenic region between yjbC and spx. Primer extension analysis uncovered a second intergenic promoter, P(3), from which transcription is elevated in cells treated with the thiol-specific oxidant diamide. P(3) is utilized by the sigma(A) form of RNA polymerase in vitro without the involvement of a transcriptional activator. Transcriptional induction from P(3) did not require an Spx-RNAP interaction and was observed in a deletion mutant lacking DNA upstream of position -40 of the P(3) promoter start site. Deletion mutants with endpoints 3' to the P(3) transcriptional start site (positions +5, +15, and +30) showed near-constitutive transcription at the induced level, indicating the presence of a negative control element downstream of the P(3) promoter sequence. Point mutations characterized by bgaB fusion expression and primer extension analyses uncovered evidence for a second cis-acting site in the P(3) promoter sequence itself. The data indicate that spx transcription is under negative transcriptional control that is reversed when disulfide stress is encountered.