We investigated the effects of a single dose of levodopa (L-dopa) on the level and extent of visual cortex activation of subjects with amblyopia and normal subjects using blood oxygenation level-dependent functional magnetic resonance imaging (fMRI). Six patients with amblyopia and 9 control patients were recruited. A baseline fMRI session was followed by a second session 90 minutes after the dose of L-dopa. Visual stimuli included vertical sinusoidal gratings with spatial frequencies of 1 and 2 cpd that were counterphased at 4 Hz. Stimuli were presented monocularly and binocularly. The fMRI response was characterized by the total volume and the average level of activation within the occipital cortex. An interocular absolute difference (IDIF) was defined in terms of said measures for between-population analysis of monocular data. After the administration of L-dopa, visual acuity improved significantly ( P = 0.026) from 0.72 +/- 0.21 (mean +/- SD) to 0.64 +/- 0.24 LogMAR in the amblyopic eye, although remaining the same in the dominant eye and in the eyes of control subjects. The response to L-dopa was found to be population-specific, as indicated by a significant treatment-by-population interaction for the volume of activation IDIF ( P = 0.018) measure: subjects with amblyopia exhibited a post-treatment increase in the volume of activation IDIF whereas control subjects showed a less prominent decrease. This post-treatment increase of IDIF in subjects with amblyopia may be explained by the decrease in the volume of activation found for the amblyopic eye after L-dopa ( P = 0.038). No L-dopa-related activation changes were detected for dominant eye or binocular stimulation in the amblyopic group, and no change was detected in control subjects. L-dopa elicits a population-specific modulation of the fMRI response, namely, a reduced total volume of activation from the amblyopic eye despite improvement in visual acuity.