The ςX and ςW extracytoplasmic function sigma factors regulate more than 40 genes in Bacillus subtilis. ςW activates genes which function in detoxification and the production of antimicrobial compounds, while ςX activates functions that modify the cell envelope. Transposon mutagenesis was used to identify loci which negatively regulate ςW or ςX as judged by up-regulation from the autoregulatory promoter site PW or PX. Fourteen insertions that activate PW were identified. The largest class of insertions are likely to affect transport. These include insertions in genes encoding two multidrug efflux protein homologs (yqgE and yulE), a component of the oligopeptide uptake system (oppA), and two transmembrane proteins with weak similarity to transporters (yhdP and yueF). Expression from PW is also elevated as a result of inactivation of at least one member of the ςW regulon (ysdB), an ArsR homolog (yvbA), a predicted rhamnose isomerase (yulE), and a gene (pksR) implicated in synthesis of difficidin, a polyketide antibiotic. In a parallel screen, we identified seven insertions that up-regulate PX. Remarkably, these insertions were in functionally similar genes, including a multidrug efflux homolog (yitG), a mannose-6-phosphate isomerase gene (yjdE), and loci involved in antibiotic synthesis (srfAB and possibly yogA and yngK). Significantly, most insertions that activate PW have little or no effect on PX, and conversely, insertions that activate PX have no effect on PW. This suggests that these two regulons respond to distinct sets of molecular signals which may include toxic molecules which are exported, cell density signals, and antimicrobial compounds.