IMPORTANCE: Aquaporin 4 antibody (AQP4-Ab)-negative patients with longitudinally extensive transverse myelitis (LETM) behave differently from those with AQP4-Ab. Aquaporin 4 antibody-negative neuromyelitis optica (NMO) is rare when good assays are used. OBJECTIVE: To assess if AQP4-Ab-negative patients with LETM share similar disease characteristics with AQP4-Ab-positive patients or whether they have distinct features and alternative diagnoses. DESIGN We collated clinical and paraclinical data on patients with LETM identified through the Oxford NMO clinical database. Aquaporin 4 antibodies were tested using 2 sensitive assays. We describe the features of patients with LETM, compare findings between patients with and without AQP4-Ab, and describe alternative diagnoses in AQP4-Ab-negative patients. SETTING: Single specialist UK center for NMO. PARTICIPANTS: Seventy-six adult patients with LETM. MAIN OUTCOMES AND MEASURES: Comparison of clinical and paraclinical data. RESULTS: Fifty-eight percent of patients were AQP4-Ab positive. Alternative diagnoses could usually be identified in AQP4-Ab-negative patients, including those fulfilling NMO diagnostic criteria. Only 6.5% of patients had "true" seronegative NMO and 6.5% had idiopathic LETM. There were some important differences between AQP4-Ab-positive and -negative cases, including older onset age, higher proportion of females, lower incidence of simultaneous optic neuritis, lower frequency of conus involvement, and higher prevalence of coexisting autoimmune disorders in AQP4-Ab-positive cases. Attack severity and degree of recovery were similar in the 2 groups. CONCLUSIONS AND RELEVANCE: Patients with LETM without AQP4-Ab include a number of different diagnostic categories and it is not surprising therefore that they show important differences compared with AQP4-Ab-positive patients, even when considering only those fulfilling current NMO diagnostic criteria. Thus, we suggest that diagnoses such as myelin-oligodendrocyte glycoprotein antibody disease, multiple sclerosis, acute disseminated encephalomyelitis, and postinfectious disorders should be exclusions in the NMO diagnostic criteria and AQP4-Ab-positive and antibody-negative NMO/NMO spectrum disorder cohorts should be analyzed separately.