In vivo imaging of adenosine 2A receptors (A2A) in the brain has attracted significant interest from the scientific community, because studies have shown that dysregulation of these receptors is implicated in a variety of neurodegenerative and psychiatric disorders, including Parkinson and Huntington diseases. This work aimed to describe the kinetic properties, test-retest results, and dosimetry estimates of (123)I-MNI-420, a SPECT radiotracer for the in vivo imaging of A2A in the brain. METHODS: Nine healthy human subjects were enrolled in this study; 7 completed (123)I-MNI-420 brain SPECT studies, and 2 participated in whole-body planar imaging evaluating (123)I-MNI-420 biodistribution and dosimetry. For 3 of the brain SPECT studies, arterial blood was collected for invasive modeling. Noninvasive models were also explored, including Logan graphical analysis and simplified reference tissue models. Test-retest reliability was assessed in 4 subjects. To evaluate radiotracer biodistribution and dosimetry, serial whole-body images were acquired immediately after injection and at selected time points after injection. Urine samples were collected over a period of 21 h to calculate urinary excretion. RESULTS: (123)I-MNI-420 rapidly entered the human brain and displayed uptake consistent with known A2A densities. At pseudoequilibrium (reached at 90 min after radiotracer injection), stable target-to-cerebellum ratios of around 1.4-2.0 were determined. Binding potentials around 0.8-1.2 were estimated using different kinetic models and the cerebellum as the reference region. Average test-retest variability in the striatum was 4.8%, 3.5%, and 6.5% for the simplified reference tissue model, Logan graphical analysis, and standardized uptake value ratio methods, respectively. The estimated radiation effective dose determined from whole-body studies was 0.036 mSv/MBq. CONCLUSION: The data indicate that (123)I-MNI-420 is a useful SPECT radiotracer for imaging A2A in the brain and has radiation doses that would allow for multiple scans in the same research subject each year. The availability of (123)I-MNI-420 offers the possibility of investigating A2A activity in specific conditions and evaluating drug occupancy for A2A candidate therapeutics.