BACKGROUND AND PURPOSE: Nanobodies are promising antigen-binding moieties for molecular imaging and therapeutic purposes due to their favorable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the central nervous system (CNS) remains to be demonstrated. EXPERIMENTAL APROACH: We have assessed the blood-brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei Variant-specific Surface Glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using 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. KEY RESULTS: The combined read-out methodologies demonstrate that disposition of Nb_An33 can be detected in the brain of healthy rats following intravenous injection and that inflammation-induced damage to the blood-brain barrier significantly increases the nanobody perfusion efficiency. We also 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. The presented data also suggest that fast blood clearance might hamper efficient targeting of specific nanobodies to the CNS. CONCLUSIONS AND IMPLICATIONS: Nanobodies can perfuse into the brain parenchyma, especially in pathological conditions where the blood-brain barrier integrity is compromised.