Background: Botulinum neurotoxin A (BoNT-A) is a highly neurotoxic drug and frequently used in patients. Knowledge on the optimal way of administration of BoNT-A and its subsequent distribution is still rather limited. An accurate method for monitoring these processes might be the use of radiolabelled BoNT-A. In this paper, we report our feasibility study on labelling BoNT-A with high-dose iodine-125 (125I) via IODOGEN-coated BoNT-A method. Methods: Using cetuximab as model substrate for BoNT-A, a miniaturization of the IODOGEN-coated mAb method was developed with special attention to the minimum required amount of the oxidant IODOGEN, while the amount of substrate, reaction volume and reaction time were downsized. Labelling efficiency and radiochemical purity were determined by TLC, integrity by SDS-PAGE and HPLC and immunoreactivity by cell-binding assay. BoNT-A (50 μg) was labelled with 125I by coating with 2.5 μg IODOGEN, in a total reaction volume of 250 μL and a reaction time of 90 s. 125I-BoNT-A was purified by size exclusion chromatography (PD10 column) using ascorbic acid solution (5 mg/ml, pH = 5) as eluent. Quality analysis of 125I-BoNT-A was performed by an in vitro bladder strip model, an electrochemiluminescence assay and an Endopep assay. Results: Cetuximab (50 μg) labelling with 125I (15 to 150 MBq) resulted in a labelling efficiency of 70% to 80%, a radiochemical purity of >99%, an immunoreactivity of >95% and a retained integrity on SDS; HPLC analysis revealed partly affected integrity when 110 to 150 MBq 125I was used, i.e. when the averaged I/mAb molar ratio exceeded 3. Addition of HEPES (20 mM) and lactose (1.25%) (lyophilized BoNT-A contains HEPES and lactose) decreased the labelling efficiency to 44% to 54%. BoNT-A (50 μg) labelling with 125I (97.2 to 98.3 MBq) resulted in labelling efficiency of 51% to 52% with a radiochemical purity >98.5%, a specific activity of 150.5 to 152.9 MBq/nmol and an I/BoNT-A molar ratio of 1.86 to 1.90. The in vitro bladder strip model showed no bioactivity of 125I-BoNT-A when compared to unlabelled BoNT-A. The electrochemiluminescence and Endopep assay demonstrated around 10% and 15% bioactivity of 125I-BoNT-A compared to unlabelled BoNT-A, respectively. The remaining bioactivity correlates within the Poisson distribution with the amount of BoNT-A molecules that does not bear an iodine atom. Conclusions: BoNT-A was successfully radio-iodinated with an activity high enough to enable in vivo measurement of nanograms of BoNT-A, which could be used in studying optimization of administration techniques of BoNT-A. The bioactivity of a BoNT-A molecule is, however, lost upon the introduction of an iodine atom into the tyrosine moiety of this sensitive molecule. © 2015, van Uhm et al.; licensee Springer.