Neuromyelitis optica (NMO) is thought to be caused by immunoglobulin G autoantibodies (NMO‐IgG) against astrocyte water channel aquaporin‐4 (AQP4). A recent study (Hinson et al. (2012) Proc Natl Acad Sci USA 109:1245‐1250) reported that NMO‐IgG inhibits AQP4 water permeability directly and causes rapid cellular internalization of the M1 but not M23 isoform of AQP4, resulting in AQP4 clustering, enhanced complement‐dependent cytotoxicity, and tissue swelling. Here, we report evidence challenging this proposed mechanism of NMO‐IgG‐mediated pathology. We measured osmotic water permeability by stopped‐flow light scattering on plasma membrane vesicles isolated from AQP4‐expressing CHO cells, an approach that can detect changes in water permeability as small as 5% and is not confounded by internalization effects. We found similar single‐molecule water permeability for M1‐AQP4 tetramers and M23‐AQP4 clusters (orthogonal arrays of particles, OAPs). Exposure of AQP4 to high concentrations of NMO‐IgG from six seropositive NMO patients, and to high‐affinity recombinant monoclonal NMO antibodies, did not reduce AQP4 water permeability. Also, NMO‐IgG did not reduce water permeability in AQP4‐reconstituted proteoliposomes. In transfected cells expressing M1‐ or M23‐AQP4 individually, NMO‐IgG caused more rapid internalization of M23‐ than M1‐AQP4. In cells coexpressing both isoforms, M1‐ and M23‐AQP4 comingled in OAPs that were internalized together in response to NMO‐IgG. Super‐resolution imaging and native gel electrophoresis showed that the size of AQP4 OAPs was not altered by NMO sera or recombinant NMO antibodies. We conclude that NMO‐IgG does not: (i) inhibit AQP4 water permeability, (ii) cause preferential internalization of M1‐AQP4, or (iii) cause intramembrane AQP4 clustering. © 2012 Wiley Periodicals, Inc.