Bacteria respond to diverse growth-limiting stresses by producing a large set of general stress proteins. In Bacillus subtilis and related Gram-positive pathogens, this response is governed by the σB transcription factor. To establish the range of cellular functions associated with the general stress response, we compared the transcriptional profiles of wild and mutant strains under conditions that induce σB activity. Macroarrays representing more than 3900 annotated reading frames of the B. subtilis genome were hybridized to 33P-labelled cDNA populations derived from (i) wild-type and sigB mutant strains that had been subjected to ethanol stress; and (ii) a strain in which σB expression was controlled by an inducible promoter. On the basis of their significant σB-dependent expression in three independent experiments, we identified 127 genes as prime candidates for members of the σB regulon. Of these genes, 30 were known previously or inferred to be σB dependent by other means. To assist in the analysis of the 97 new genes, we constructed hidden Markov models (HMM) that identified possible σB recognition sequences preceding 21 of them. To test the HMM and to provide an independent validation of the hybridization experiments, we mapped the σB-dependent messages for seven representative genes. For all seven, the 5′ end of the message lay near typical σB recognition sequences, and these had been predicted correctly by the HMM for five of the seven examples. Lastly, all 127 gene products were assigned to functional groups by considering their similarity to known proteins. Notably, products with a direct protective function were in the minority. Instead, the general stress response increased relative message levels for known or predicted regulatory proteins, for transporters controlling solute influx and efflux, including potential drug efflux pumps, and for products implicated in carbon metabolism, envelope function and macromolecular turnover.