Our study was undertaken to investigate whether beta2-glycoprotein I (GPI) is relevant for enhancing anticardiolipin antibody (aCL) binding in stroke patients, in particular view of vascular risk factors and recurrence of previous cerebral ischemic events. One-hundred and twenty-one sera from patients with ischemic stroke and 154 control sera from patients with non-ischemic neurological disorders (n = 43) and healthy subjects (n = 111) were included in the study. All sera were tested for either GPI-independent aCL and GPI-dependent aCL. GPI-independent aCL were detected in two (1.7%) stroke patients. When GPI was added to the assay system, 13 (10.8%) sera were positive. Of the 43 sera in the neurological control group one was positive for both GPI-independent aCL and GPI-dependent aCL. Multiple linear regression in the stroke group revealed that GPI-dependent aCL are marginally associated with sex, prior TIA/strokes and embolism. More importantly, GPI-dependent aCL were significantly more frequent in stroke patients with a history of prior TIA/strokes compared with patients with a first ischemic stroke (p = 0.029). The present study demonstrates that aCL in stroke patients are of the GPI-dependent type and emphasizes the importance of adding GPI in the immunoassay for the optimal detection of aCL. Furthermore, it supports the recommendation of testing aCL in those patients who experience unexplained recurrent cerebral ischemic events.