Rotating bending fatigue tests were conducted on three sets of quench and tempered 4140 steel samples, representing three distinctly different inclusion populations: high oxygen/low sulfur, low oxygen/high sulfur, and low oxygen/low sulfur. The inclusion populations for the three sample sets were characterized using both a scanning electron microscope-based image analysis system, primarily for the micro-inclusions, and a high-resolution ultrasonic system for the macro-inclusions. All three sample sets were evaluated using both longitudinally and transverse-oriented specimens in the rotating bending tests. The transverse samples displayed significantly lower fatigue performance (approximately 50 % lower fatigue strength values) than the longitudinal samples. Furthermore, the high sulfur sample set clearly had the lowest performance in the transverse orientation. Although there was more scatter with the data on the longitudinal samples, the high oxygen sample set had a lower fatigue strength and a higher percentage of failures initiating at subsurface oxides than the other two sample sets.