Isotachophoretic separations are triggered at the border of a nanochannel-induced ion-depleted zone. This depletion zone acts as a terminating electrolyte and is created by concentration polarization over the nanochannel. We show both continuous and discrete sample injections as well as separation of up to four analytes. Continuous injection of a spacer compound was used for selective analyte elution. Zones were kept focused for over one hour, while shifting less than 700 μm. Moreover, zones could be deliberately positioned in the separation channel and focusing strength could be precisely tuned employing a three-point voltage actuation scheme. This makes depletion zone isotachophoresis (dzITP) a fully controllable single-electrolyte focusing and separation technique. For on-chip electrokinetic methods, dzITP sets a new standard in terms of versatility and operational simplicity.