The mechanism of inhibition by cytoplasmic nickel of slowly activating channels in radish vacuoles was investigated using the patch-clamp technique. The decrease in the macroscopic current induced by the presence of nickel in the cytoplasmic solution can be described by a Michaelis-Menten equation with an apparent dissociation constant of 0.45+/-0.03 mM. At the single-channel level, nickel moderately decreases the single-channel conductance, since the ratio between the chord conductance in the presence and in the absence of 1 mM cytosolic nickel is 0.89+/-0.06. Experiments performed to study the interaction between calcium, an activator of the channel, and nickel strongly suggest that these two ions bind to two distinct molecular sites. A simple mathematical model predicting the experimental observations is presented.