Purpose: This study investigates interocular differences in the level and extent of cortical activation in amblyopic and normal subjects. Subjects and Methods: Blood oxygenation level dependent functional magnetic resonance imaging (FMRI) was performed at 1.5 T. A total of 5 subjects with amblyopia and 6 control subjectswere recruited. Visual stimuli included a homogeneous field flickering at 8 Hz and vertical sinusoidal gratings with spatial frequencies of 0.5, 1, and 2 cycles per degree of visual angle (counterphased at 8 Hz). Baseline images were taken in darkness. Stimuli were presented monocularly and binocularly. Data analysis was constrained to the occipital cortex, including striate and extrastriate areas. An absolute percent difference measure defined interocular differences in terms of total area, average level of activation, and a pooled activation parameter. Results: Subjects with amblyopia exhibited a significantly larger (P <.05) interocular activation difference compared to controls. The level of activation driven by monocular stimulation of the amblyopic eye was reduced by 8.25% ± 1.3% relative to the dominant eye. Controls showed an interocular difference in level of activation of 4.82% ± 0.74%. The total area of activation driven by the amblyopic eye was reduced by 34.86% ± 6.25% relative to the dominant eye. Controls showed an interocular difference in total area of activation of 20.80% ± 3.67%. Subjects with amblyopia also manifested significantly greater (P <.05) cortical area and level of activation differences between the binocular and monocular states; the dominant eye response differed less from the binocular response than did the amblyopic eye response. Conclusion: FMRI is sensitive to amblyopia-related deficits in the human occipital cortex and, therefore, has potential as a tool for basic amblyopia research. (J AAPOS 2002;6:300-8)