Background and Purpose— Antibodies against neuronal antigens develop in patients after stroke and some may serve as biomarkers of neuronal injury. We aimed to determine whether antibodies against subunit 1 (GluN1) of the N -methyl- d -aspartate receptor also develop after stroke and if so, whether they correlate with stroke characteristics. Methods— Forty-eight patients with ischemic stroke and 96 healthy controls were tested for the presence of serum antibodies targeting GluN1. Testing was conducted using 20-kDa recombinant GluN1-S2 peptide (by ELISA and Western blotting) and on rat brain tissue (by Western blotting and immunohistochemistry). Clinical examinations and computed tomographic brain scans were performed to assess clinical state and infarct size and location. Results— Of the 48 patients with ischemic stroke, 21 (44%) had antibodies that reacted with the recombinant GluN1-S2. There was no evidence of antibody binding to intact GluN1 in brain tissue. Western blot appearances suggested reactivity with GluN1 degradation products. Patients with anti–GluN1-S2 antibodies were more likely to have higher National Institutes of Health Stroke Scale scores, larger infarcts, and more frequent cortical involvement. Of the 96 controls, only 3 (3%), all aged >50 years, had antibodies that reacted with GluN1-S2 at low levels. Conclusions— Antibodies that bind recombinant GluN1-S2 peptides (but not the intact GluN1 protein) develop transiently in patients after stroke in proportion to infarct size, suggesting that these antibodies are raised secondarily to neuronal damage. The anti–GluN1-S2 antibodies may provide useful information about the presence and severity of cerebral infarction. This will require confirmation in larger studies.