Neuropsychological investigations of patients with Parkinson's disease, schizophrenia, or attention deficit disorder converge with psychopharmacological studies in animals and healthy volunteers to implicate dopamine (DA) pathways in timing. In parallel, single-cell recording and functional neuroimaging studies have highlighted the importance of basal ganglia, prefrontal cortex, and supplementary motor area (SMA) for timing. In a placebo-controlled, within-subject design, we combined event-related functional magnetic resonance imaging with a DA manipulation (acute phenylalanine/tyrosine depletion; APTD) in healthy volunteers to pinpoint the neuroanatomical and functional substrates of the DA modulation of timing. Behaviorally, APTD selectively impaired accuracy of perceptual timing, with no effect on performance of a color-control task matched for difficulty, working memory (WM), and attentional demands. Neurally, APTD attenuated timing-specific activity in the putamen and SMA. Notably, APTD-induced decreases in brain activity were directly correlated to APTD-induced impairments in timing performance. Moreover, APTD modulated timing-specific activity selectively during initial storage of the sample duration, but had no effect during its subsequent retrieval or comparison to a probe. Our results do not simply reflect DA modulation of WM since the color task controlled for the WM updating process necessary for timing of durations in the seconds range. Moreover, preliminary evidence indicated APTD effects on putamen and SMA were greater for subsecond (540 ms) than suprasecond (1080 ms) durations, when WM demands would actually be lower. Instead, we show for the first time in healthy humans that DA manipulation perturbs timing by attenuating the activity in putamen and SMA that mediates initial storage of temporal information into WM.