Subject classification: 61.72.Ji; 62.20.Mk; 78.70.Bj; S1.1Positron annihilation lifetime and Doppler broadening measurements have been performed to study the early stage of fatigue in type 304 stainless steel at various positions of samples at room temperature. Both the average lifetime of positron and the S parameter have increased with increasing number of fatigue cycles at each position. From two-lifetime component analysis, the component of the longer lifetime of about 170 ps, corresponding to single or di-vacancies, has been observed from 0.01% Nf fatigue, and it has remained stable until 10% Nf of the fatigue life. However, the lifetime has increased rapidly to about 230 ps toward a fatigue life of about 20% Nf at a certain position of the sample. Vacancy clusters have possibly been formed to some extent at this fatigue life. Moreover, the fatigue failure has occurred at almost the same position as the one where the longer positron lifetime was observed. These results suggest that the generation of micro cracks during fatigue test is related to the formation of three-dimensional vacancy clusters. Detailed measurements of positron lifetime and Doppler broadening, have also been carried out for small areas of fractured samples. From these experiments, it has been found that stress concentration occurred at a central position of a sample during fatigue and that this can be probed by the increase of the positron lifetime.