Amblyopia, sometimes called "lazy eye," is a relatively common developmental visual disorder well characterized behaviorally; however, the neural substrates associated with amblyopia in humans remain unclear. We hypothesized that abnormalities in the cerebral cortex of subjects with amblyopia exist, possibly as a result of experience-dependent neuronal plasticity. Anatomic magnetic resonance imaging (MRI) and psychophysical vision testing was carried out on 74 subjects divided into two age ranges, 7-12 years and 18-35 years, and three diagnoses, strabismic amblyopia, anisometropic amblyopia, and normal vision. We report a behavioral impairment in contrast sensitivity for subjects with amblyopia, consistent with previous reports. When the high-resolution MRI brain images were analyzed quantitatively with optimized voxel-based morphometry, results indicated that adults and children with amblyopia have decreased gray matter volume in visual cortical regions, including the calcarine sulcus, known to contain primary visual cortex. This finding was confirmed with a separate region-of-interest analysis. For the children with amblyopia, additional gray matter reductions in parietal-occipital areas and ventral temporal cortex were detected, consistent with recent reports that amblyopia can result in spatial location and object processing deficits. These data are the first to provide possible neuroanatomic bases for the loss of binocularity and visual sensitivity in children and adults with amblyopia.