Exposure of bacteria to diverse growth-limiting stresses induces the synthesis of a common set of proteins which provide broad protection against future, potentially lethal stresses. Among Bacillus subtilis and its relatives, this general stress response is controlled by the sigmaB transcription factor. Signals of environmental and energy stress activate sigmaB through a multicomponent network that functions via a partner switching mechanism, in which protein-protein interactions are governed by serine and threonine phosphorylation. Here, we tested a central prediction of the current model for the environmental signaling branch of this network. We used isoelectric focusing and immunoblotting experiments to determine the in vivo phosphorylation states of the RsbRA and RsbS regulators, which act in concert to negatively control the RsbU environmental signaling phosphatase. As predicted by the model, the ratio of the phosphorylated to unphosphorylated forms of both RsbRA and RsbS increased in response to salt or ethanol stress. However, these two regulators differed substantially with regard to the extent of their phosphorylation under both steady-state and stress conditions, with RsbRA always the more highly modified. Mutant analysis showed that the RsbT kinase, which is required for environmental signaling, was also required for the in vivo phosphorylation of RsbRA and RsbS. Moreover, the T171A alteration of RsbRA, which blocks environmental signaling, also blocked in vivo phosphorylation of RsbRA and impeded phosphorylation of RsbS. These in vivo results corroborate previous genetic analyses and link the phosphorylated forms of RsbRA and RsbS to the active transmission of environmental stress signals.