Using ion-selective microelectrodes, the basis of Nod factor-induced changes in the plasma membrane potential was analysed by measuring the extracellular free concentrations of Ca2+, K+, H+ and Cl– in the root hair zone of alfalfa. After addition of the Rhizobium meliloti Nod factor NodRm-IV(C16:2,S) at a concentration of 0.1 μM, a decrease in [Ca2+] was observed first, which was followed after a few seconds by an increase of [Cl–], by an alkalinization, and then by a delayed increase of [K+], all of which were transient changes. Simultaneously with the appearance of Cl– ions in the root hair zone, a decrease in cytosolic [Cl–] was measured. It was concluded that the depolarization was caused by temporary short-circuiting of the proton pump through the rapid release of Cl– ions along their steep electrochemical gradient. Since under resting conditions the driving force for K+ ions was inwardly directed, their release was delayed until their driving force was inverted. This indicates that K+ serves as a charge balance that eventually stops depolarization and initiates repolarization. Since the decrease in [Ca2+] was observed seconds before the increase in [Cl–] and the depolarization, it is argued that Ca2+ entering into the cell does not cause the depolarization directly, but might initiate it by triggering the activation of an anion channel that then releases the chloride ions. The observations that the Ca2+ ionophore A23187 mimicks the Nod factor response, and that the Ca2+ channel antagonist nifedipine inhibits this response, support the idea that Ca2+ plays a primary role in the transduction of the Nod signal in alfalfa.