Resistance against antimicrobial peptides in many Gram-positive bacteria is mediated by detoxification systems that are comprised of a two-component regulatory system and an ATP-binding-cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for detection of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. We have taken a multi-disciplinary approach combining experimental and computational methodology to unravel the mechanistic details of this unique signaling pathway, exemplified by the bacitracin resistance system BceRS-BceAB of Bacillus subtilis. We show that the transporters and kinases form a sensory complex in the membrane, and that the histidine kinase directly responds to changes in the activity of the transporter. This appears to occur via complete control of kinase conformation by the transporter, which to our knowledge is unprecedented among bacterial signaling systems. We propose that this novel regulatory strategy effectively implements a flux-sensing mechanism, allowing the cell to adjust the rate of de novo transporter synthesis to precisely the level needed for protection.