The utility of three-dimensional anisotropy contrast (3DAC) magnetic resonance (MR) axonography, a method sensitive to neuronal fibers and their directionality, was investigated in the clinical setting using a 3-tesla MR imaging system based on a General Electric Signa platform. The study focused on healthy volunteers and patients with common structural central nervous system disorders, namely chronic infarction, brainstem cavernous hemangioma, supratentorial meningioma, and astrocytoma. Three orthogonal anisotropic diffusion-weighted images were first obtained. Three primary colors were each assigned to a diffusion-weighted image, respectively, and the images were subsequently combined into a single-color image in full-color spectrum (3DAC MR axonography image). Fiber-tract definition in the cerebral peduncle of the midbrain of healthy volunteers showed intersubject variation, with two general patterns recognized: dispersed (60% of cases) and compact (40% of cases). Pathological alterations in the fiber tracts were readily identified in cases involving wallerian degeneration of the pyramidal tract, as illustrated in the cases of chronic infarction. Displacement of major tracts, such as the medial lemniscus or corticospinal tract, as well as fiber directionality, was also easily recognized in cases of mass lesions. As an imaging method uniquely capable of providing information regarding axonal connectivity, 3DAC MR axonography appears to have promising potential for routine clinical application.