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Melatonin is a neurohormone that modulates several physiological functions in mammals through the activation of melatonin receptor type 1 and 2 (MT₁ and MT₂). The melatonergic system is an emerging therapeutic target for new pharmacological interventions in the treatment of sleep and mood disorders; thus, imaging tools to further investigate its role in the brain are highly sought-after. We aimed to develop selective radiotracers for in vivo imaging of both MT₁ and MT₂ by positron emission tomography (PET). We identified four previously reported MT ligands with picomolar affinities to the target based on different scaffolds which were also amenable for radiolabeling with either carbon-11 or fluorine-18. [¹¹C]UCM765, [¹¹C]UCM1014, [¹⁸F]3-fluoroagomelatine ([¹⁸F]3FAGM), and [¹⁸F]fluoroacetamidoagomelatine ([¹⁸F]FAAGM) have been synthesized in high radiochemical purity and evaluated in wild-type rats. All four tracers showed moderate to high brain permeability in rats with maximum standardized uptake values (SUV_max of 2.53, 1.75, 3.25, and 4.47, respectively) achieved 1-2 min after tracer administration, followed by a rapid washout from the brain. Several melatonin ligands failed to block the binding of any of the PET tracer candidates, while in some cases, homologous blocking surprisingly resulted in increased brain retention. Two ¹⁸F-labeled agomelatine derivatives were brought forward to PET scans in non-human primates and autoradiography on human brain tissues. No specific binding has been detected in blocking studies. To further investigate pharmacokinetic properties of the putative tracers, microsomal stability, plasma protein binding, log D, and membrane bidirectional permeability assays have been conducted. Based on the results, we conclude that the fast first pass metabolism by the enzymes in liver microsomes is the likely reason of the failure of our PET tracer candidates. Nevertheless, we showed that PET imaging can serve as a valuable tool to investigate the brain permeability of new therapeutic compounds targeting the melatonergic system.