Positron annihilation lifetime and Doppler broadening measurements have been performed to study the early stage of fatigue in type 304 stainless steels for various positions of the samples at room temperature. Both the average lifetimes of positron and the S-parameters increase with increase in the number of fatigue cycles in each position. From the two-lifetime component analysis, long lifetime components about 170 psec, which corresponds to single or di-vacancies, were observed from 0.01% fatigue, and remain stable until 10% of the fatigue life. However, the longer lifetime component increases rapidly to about 230 psec at a fatigue life of about 20% at a certain position on the sample. Vacancy cluster can be formed partially at this fatigue life. Moreover, the fatigue failure occurs at a similar position where the longer positron lifetime appeared. These results suggest that the generation of micro crack during fatigue is related to the formation of three-dimensional vacancy clusters.