Fatigue crack initiation in the polycrystalline nickel-based superalloy, René 88 DT, was investigated at 593 °C in air using an ultrasonic fatigue testing instrument. Within the testing stress range of 500 -760MPa, all fatigue failures initiated from internal regions. Large crystallographic facets were observed at the crack initiation sites in most failed fatigue samples. Critical microstructure features controlling fatigue crack initiation and the early stage of small crack growth were identified by the combination of serial sectioning, orientation imaging microscopy (OIM) and quantitative fractographic analysis. Large crystallographic facets at crack initiation sites were formed in regions close to Σ 3 annealing twin boundaries within favorably oriented large grains. The facet planes are parallel to Σ 3 annealing twin boundaries and are of {111} type. Cyclic deformation substructures were studied by transmission electron microscopy. At the low cyclic stresses and long lives examined, fatigue failure at 593°C is controlled by strain localization in large, favorably oriented grains and subsequent propagation of small cracks through neighborhoods of favorably oriented grains. Annealing twin boundaries, prevalent in the microstructure, appear to be important in cyclic strain localization.