Blood oxygenation level dependent (BOLD) contrast in skeletal may reflect the contributions of both intravascular and extravascular relaxation effects. The purpose of this study was to determine the significance of the extravascular BOLD effect in skeletal muscle at 3 T. In experiments, R 2 * was measured before and during arterial occlusion under the following conditions: (1) the leg extended and rotated (to vary the capillary orientation with respect to the amplitude of static field) and (2) with the blood's signal nulled using a multiecho vascular space occupancy experiment. In the leg rotation protocol, 3 min of arterial occlusion decreased oxyhemoglobin saturation from 67% to 45% and increased R 2 * from 34.2 to 36.6 sec 21 , but there was no difference in the R 2 * response to occlusion between the extended and rotated positions. Numerical simulations of intra-and extravascular BOLD effects corresponding to these conditions predicted that the intravascular BOLD contribution to the R 2 * change was always > 50 times larger than the extravascular BOLD contribution. Blood signal nulling eliminated the change in R 2 * caused by arterial occlusion. These data indicate that under these experimental conditions, the contribution of the extravascular BOLD effect to skeletal muscle R 2 * was too small to be practically important. Magn Reson Med 64:527-535, 2010. V C 2010 WileyLiss, Inc.Key words: mfMRI; extravascular BOLD effect; oxygenation; vessel orientation; VASO Variations in the rates of permanent and effective transverse relaxation (R 2 and R 2 *, respectively) or in T 2 -weighted signal may be used to evaluate the hemodynamic, metabolic, and structural changes associated with muscle contractions (1), an approach known as muscle functional MRI (2). During and following muscle contractions, changes in muscle R 2 and R 2 * result from increases in intracellular water content, secondary to metabolite accumulation (3,4); variations in intracellular pH (4-6); and changes in the concentrations of paramagnetic molecules such as deoxyhemoglobin, as reflected in the blood oxygenation level dependent (BOLD) effect (7,8). The magnitude of each of these changes and thus the variations in R 2 and R 2 * depend on the exercise and intensity (9), the muscle fiber type composition (3), and whole-body aerobic capacity (10). Since these factors affect R 2 and R 2 * to different degrees, quantifying the independent contributions of and interactions among these factors is important to interpret physiologic and pathologic variations in muscle R 2 and R 2 *.Regarding the source of oxygen-level dependent contrast in skeletal muscle, it is noteworthy that skeletal muscle cells themselves contain several potential magnetic field perturbers that may also contribute to susceptibility-related signal decay in skeletal muscle. However, both theoretical calculations and experimental observations suggest that a true BOLD effect, and not a muscle oxygenation level dependent effect, occurs in skeletal muscle (8). The presence of a muscle BOLD ...