The effect of microstructure on the fatigue crack initiation of fully pearlitic steels was studied through independent variation of the prior austenite grain size, pearlite colony size, and the pearlite interlamellar spacing. Increasing yield strength (controlled by decreasing the pearlite interlamellar spacing) was seen to increase the smooth and notched-bar crack initiation endurance limit. Grain and colony size variations, at constant yield strength, were seen to exhibit no effect on crack initiation. Scanning Electron Microscopy revealed smooth-bar cracks to have initiated at surface inclusions. The influence of the pearlite interlamellar spacing, reflecting a change in the effective slip length, and the differences between notched and smooth-bar fatigue specimens for studying the effects of microstructure on crack initiation are discussed.