Nanobodies are considered as very promising antigen-binding moieties for molecular imaging and therapeutic purposes due to their favorable pharmacological and pharmacokinetic properties. However, the possibility for monovalent Nanobodies to reach targets in the central nervous system (CNS) remains to be demonstrated. We have assessed the blood-brain barrier permeability for Nb_An33, a Nanobody against the Trypanosoma brucei Variant-specific Surface Glycoprotein (VSG), in healthy rats and rats that are in the encephalitic stage of African trypanosomiasis and suffer from acute brain inflammation. To assess the integrity of the blood-brain barrier, brain disposition of 2-Methoxy-IsoButyl-Isonitrile (MIBI) and Evans Blue was monitored in both experimental groups of animals. Perfusion of the different compounds was analyzed by intracerebral microdialysis, Single Photon Emission Computed Tomography (SPECT) or a combination of both methodologies. This enabled the quantification of unlabeled and (99m)Tc-labeled Nanobodies using respectively a sensitive VSG-based Nanobody-detection ELISA, radioactivity measurement in collected microdialysates and SPECT image analysis. As the ELISA analysis provided direct evidence for antigen-binding potential, this approach allowed to differentiate between active Nanobody and fragments that might be generated during the (99m)Tc-labeling process or in vivo by proteolytic processing. The combined read-out methodologies illustrate that, while brain disposition of Nb_An33 upon intravenous injection is at the limit of detection in healthy rats, inflammation-induced damage to the blood-brain barrier significantly increases (approximately 20 times) the Nanobody perfusion efficiency. Despite the enhanced brain disposition, calculated intrahippocampal Nb_An33 concentrations remained very low (131 ± 63 ng/ml with a dose of 4 mg/kg), likely resulting from the observed high clearance rates of Nanobodies in general. Collectively, we illustrate the advantage of complementing SPECT analyses with intracerebral microdialysis in brain disposition studies and suggest that it is of interest to evaluate the blood-brain barrier penetrating potential of monovalent Nanobodies in models of CNS-inflammation. However, the pharmacokinetic properties that are considered favorable for peripheral imaging and therapeutic approaches might be key factors that prelude efficient application of Nanobodies to target the CNS.