The classical dynamin family of large GTPases are ubiquitous proteins involved the interplay between the plasma membrane and the actin cytoskeleton. There are 27 known dynamin isoforms arising from alternate splicing of three dynamin genes (DNM1-3). Dynamin I (dynI) is a neuronal isoform enriched in the synapse where it performs a specialised mode of endocytosis as part of the synaptic vesicle retrieval mechanism by oligomerisation to a helix. DynI may also be involved in the maintenance of neuronal cell shape by interacting with the actin cytoskeleton and oligomerisation into ring complexes. DynI and II are alternately spliced in the middle domain, producing a and b variants of no known function. This dynamin domain is associated with self-assembly into either ring complexes or helices. Our aim was to determine the role of middle domain splice variation in dynamin I by characterising the self-assembly properties of purified recombinant dynamin isoforms. Human dynI middle domain isoforms were expressed and purified from Sf21 insect cells. The purified proteins were assayed for self-assembly using sedimentation techniques and enzyme activity using a Malachite Green colorimetric GTPase assay. DynIa has a higher basal level of GTPase activity than dyn1b. DynIb however, has increased GTPase activity in response to helix assembly around PS liposomes, indicating an higher propensity of this isoform for lipid-assisted helical assembly. The results show that two middle domain dynI isoforms assemble differently and may therefore be involved in unique cellular functions. Future research may provide evidence of a functional difference between the two isoforms in cells.