The Kdp system of Escherichia coli is composed of the high-affinity K+ transporter KdpFABC and the two regulatory proteins KdpD (sensor kinase) and KdpE (response regulator), which constitute a typical two-component system. The kdpFABC operon is induced under K+-limiting conditions and, to a lesser extent, under high osmolality in the medium. In search for the stimulus sensed by KdpD, we studied the inhibitory effect of extracellular K+ on the Kdp system at pH 6.0, which is masked by unspecific K+ transport at higher pH values. Based on KdpD derivatives carrying single aspartate replacements in the periplasmic loops which are part of the input domain, we concluded that the inhibition of the Kdp system at extracellular K+ concentrations above 5 mM is mediated via KdpD/KdpE and not due to inhibition of the K+-transporting KdpFABC complex. Furthermore, time-course analyses of kdpFABC expression revealed that a decline in the extracellular K+ concentration efficiently stimulates KdpD/KdpE-mediated signal transduction. In this report we provide evidence that the extracellular K+ concentration serves as one of the stimuli sensed by KdpD.