The effect of the preparation method and metal loading on the hydrodesulfurization (HDS) of thiophene, and on the dispersion and location of Ni and/or W species in Ni, W and NiW sulfides supported on USY zeolite, was investigated by temperature-programmed reduction (TPR), X-ray photoelectron spectroscopy, N-2 adsorption and acidity determination. The monometallic Ni catalyst prepared by ion exchange [Ni (int)] exhibited higher initial HDS activity than those prepared by impregnation [Ni (imp)] and by ion exchange and subsequent formation of small NiO clusters by NaOH treatment [Ni (clus)]. For the binary NiW catalyst series with different metal contents, the catalysts prepared by impregnating W onto the Ni (int) one showed activities comparable to those of the catalysts prepared by impregnating Ni onto the W/USY sample. These catalyst series showed much higher activities than those prepared by impregnating W onto the Ni (clus). The characterization results revealed that catalysts displayed significant differences in dispersion, location and sulfidation of the Ni and W phases dependent on the preparation method. However, none of these factors alone accounted for the activity trend. In general, it was found that a combination of high acidity and metal dispersion led to catalysts with high initial HDS activity.