In this paper the effect of small defects on fatigue threshold of different series of nitrided and nitride-shot peened low alloy steel specimens is investigated by means of experimental rotating bending tests on sandglass specimens. Micro-holes acting as pre-cracks are introduced by means of different methods (electro-erosion and indentation) in order to evaluate the effect of defect size as well as the influence of the process to generate them on the fatigue threshold. The results, interpreted in terms of ΔK th , show good agreement with estimated ones, calculated by a formula that considers the experimentally measured residual stresses, FWHM parameter, micro-hardness values and SEM observation of broken and run-out specimens. A critical discussion of the obtained results highlights the influence of the defect size and of the method used to generate the micro-holes on the fatigue threshold.