Bacillus subtilis responds to signals of environmental and metabolic stress by inducing over 40 general stress genes under the control of the sigma B transcription factor. sigma B activity is regulated post-translationally by a multi-component network composed of two coupled partner-switching modules, RsbX-RsbS-RsbT and RsbU-RsbV-RsbW, each containing a serine phosphatase (X or U), an antagonist protein (S or V), and a switch protein/serine kinase (T or W). The upstream module (X-S-T) is required to transmit signals of environmental stress. In contrast, the downstream module (U-V-W) is required to transmit signals of energy stress as well as the environmental signals conveyed to it from the upstream module. Until now the function of the rsbR gene product was unknown. RsbR shares significant sequence similarity with the RsbS and RsbV antagonist proteins whose phosphorylation states control key protein-protein interactions within their respective modules. Here we present evidence that RsbR is associated with RsbS in the upstream, environmental-sensing module. To investigate RsbR function, we constructed deletion and point mutations within rsbR and tested their effects on expression of sigma B-dependent reporter fusions, both singly and in combination with other rsb mutations. To determine the possible interaction of RsbR with other Rsb proteins, we tested the ability of wild-type or mutant RsbR to activate transcription in the yeast two-hybrid system in conjunction with other Rsb regulators. On the basis of this genetic analysis, we conclude that RsbR is a positive regulator which modulates sigma B activity in response to salt and heat stress. Our data further suggest that: (i) RsbR influences the antagonist function of RsbS by direct protein-protein interaction; and (ii) this interaction with RsbS is likely controlled by the phosphorylation state of RsbR.