The sigX gene, identified as part of the international effort to sequence the Bacillus subtilis genome, has been proposed to encode an alternative sigma factor of the extracytoplasmic function (ECF) subfamily. The sigX gene is cotranscribed with a downstream gene, ypuN, during logarithmic and early stationary phases of growth. We now report that strains lacking sigma(X) are impaired in the ability to survive at high temperature whereas a ypuN mutant has increased thermotolerance. We overproduced and purified sigma(X) from Escherichia coli and demonstrate that in vitro, both sigma(A) and sigma(X) holoenzymes recognize promoter elements within the sigX-ypuN control region. However, they have distinct salt optima such that sigma(A)-dependent transcription predominates at low salt while sigma(X)-dependent transcription predominates at high salt. A 54-bp region upstream of sigX suffices as a sigma(X)-dependent promoter in vivo, demonstrating that sigX is at least partially under positive autoregulatory control. Mutation of ypuN increases expression from the sigma(X)-dependent promoter in vivo, suggesting that ypuN may encode a negative regulator of sigma(X) activity.