Cellular processes require specific interactions between cognate protein partners and concomitant discrimination against noncognate partners. Signal transduction by classical two-component regulatory systems typically entails an intermolecular phosphoryl transfer between a sensor kinase (SK) and a cognate response regulator (RR). Interactions between noncognate partners are rare because SK/RR pairs coevolve unique interfaces that dictate phosphotransfer specificity. Here we report that the in vitro phosphotransfer specificity is relaxed in hybrid two-component systems (HTCSs) from the human gut symbiont Bacteroides thetaiotaomicron, which harbor both the SK and RR in a single polypeptide. In contrast, phosphotransfer specificity is retained in classical two-component regulatory systems from this organism. This relaxed specificity enabled us to rewire a HTCS successfully to transduce signals between noncognate SK/RR pairs. Despite the relaxed specificity between SK and RRs, HTCSs remained insulated from cross-talk with noncognate proteins in vivo. Our data suggest that the high local concentration of the SK and RR present in the same polypeptide maintains specificity while relaxing the constraints on coevolving unique contact interfaces.