MION-46L, a superparamagnetic iron oxide contrast agent, was investigated for its ability to increase the sensitivity of in vivo 3D MRI in the detection of brain lesions in a chronic experimental allergic encephalomyelitis (crEAE) mouse model. Lesion conspicuity on postcontrast 3D MRI was dramatically enhanced as compared to precontrast images corresponding to areas of inflammatory and demyelinating lesions. MION-46L could be detected on Prussian blue iron stain in the vascular endothelium, the perivascular space, and in macrophages within perivascular cuffs and areas of inflammation and demyelination. By taking advantage of the MION-46L induced macroscopic susceptibility effect, acute early lesions measuring only 100 microm in diameter could be detected. MION-46L enhanced MRI may be used to 1) provide a unique sensitivity in EAE lesion detection and correlate imaging to histopathology; 2) help to understand EAE lesion development and its underlying pathophysiology; and 3) eventually assist in preclinical screening of new experimental therapies directed at patients with multiple sclerosis (MS).