Dopamine D3 receptors are preferentially localized in the limbic system and midbrain, and thus may be involved in the pathophysiology of neuropsychiatry disorders. [11C](+)-PHNO is the first preferential D3 receptor radioligand in humans, yet there are no blockade studies with a D3 receptor antagonist in humans. This study characterized the blockade of [11C](+)-PHNO binding by ABT-925, a D3 receptor antagonist, in healthy male subjects. Sixteen subjects underwent 2-3 positron emission tomography (PET) scans, at baseline and following one or two doses of ABT-925 ranging from 50 mg to 600 mg. Receptor occupancies were estimated for globus pallidus, substantia nigra, caudate, putamen, and ventral striatum. At the 600-mg dose (n=9), ABT-925 receptor occupancy (mean+/-s.d.) was higher in substantia nigra (75+/-10%) and globus pallidus (64+/-22%) than in ventral striatum (44+/-17%), caudate (40+/-18%) and putamen (38+/-17%) (ANOVA: F4,140=15.02, p<0.001). The fractions of [11C](+)-PHNO binding attributable to D3 receptors in D3 receptor-rich regions were 100% (substantia nigra) and 90% (globus pallidus), and in D2 receptor-rich regions were 55% (caudate) and 53% (putamen). The ED50 of ABT-925 was 4.37 microg/ml across regions. Our results demonstrate that [11C](+)-PHNO binding can be blocked by a D3 receptor antagonist and confirm preclinical findings that [11C](+)-PHNO signal in the substantia nigra and globus pallidus is mainly reflective of its binding to D3 receptors. Thus, [11C](+)-PHNO seems a suitable PET radiotracer to estimate D3 receptor occupancy in humans.