The effect of the calcination temperature on the evolution of the active phase in NiW/gamma-Al2O3 catalysts during sulfidation has been studied using high resolution transmission electron microscopy (HREM) and temperature programmed sulfidation (TPS). The evolution of the active phase is discussed in view of the performance of the catalysts in the hydrodesulfurization (HDS) of thiophene. Dependent on the pretreatment, three different species were observed with HREM : (i) subnanometer clusters (about 0.5 nm diameter), (ii) nanometer size particles (1 to 2 mn diameter), and (iii) WS2-like slabs (2-3 nm length). The clusters and particles predominantly appear after low temperature sulfidation of catalysts calcined at 673 and 823 K. Hence, it is concluded that they are initially formed from the oxidic catalyst precursor. WS2-like slabs are predominantly formed during high temperature sulfidation, independent of the calcination temperature. Activity measurements for the HDS of thiophene show that all phases exhibit a significant catalytic activity. HREM and activity measurements on various freshly sulfided and spent catalysts showed no significant differences in the catalysts prior to and after the reaction.