A new two-step potential waveform for pulsed amperometric detection (PAD) of carbohydrates at polycrystalline gold working electrode is described. In contrast to standard triple- and quadruple-potential waveforms, in the two-step potential waveform cathodic electrode conditioning/re-activation is applied exclusively. Waveform consists of cathodic re-activation potential in hydrogen evolution region (from -1.4 V to -2.0 V vs. Ag/AgCI) and detection potential at +0.2 V. The cathodic processes responsible for activation of Au electrode in alkaline medium were characterized by fast scan cyclic voltammetry, chronoamperometry, and chronocoulometry. It was shown that application of the short negative re-activation potential maintains the electrode surface clean and active and completely substitutes oxidative cleaning/activation at potentials higher than +0.3 V used in PAD previously. Both desorptive reduction of the AuOH(ads) and hydrogen evolution reaction play a crucial role in effective cleaning/regeneration of the Au electrode in pulsed sequence. The two-step waveform preserves all analytical properties of three- and four-step potential waveforms and does not result in fouling or dissolution of Au electrode surface in a prolonged use. Square wave sequence substantially simplifies pulsed amperometric detection and enables high frequency sampling required for FIA and capillary IC systems. (C) 2011 Elsevier B.V. All rights reserved.