Prolactin (PRL) is closely associated with autoimmune diseases in animal models and humans, and several disease-related autoantibodies were reported in increased titers in patients with hyperprolactinemia (HPRL). We studied the presence of anti-endothelial cell antibodies (AECA) and other autoantibodies in sera of female patients with HPRL. Sera from 25 HPRL patients and 10 healthy female controls were tested for AECA (against both macrovascular and microvascular endothelial cell antigens), anti-dsDNA, and anti-cardiolipin (anti-CL) using ELISA. Sera were considered positive for the autoantibody when the optical density (OD) value was more than 3 s.d. above the mean of the OD in normal controls. Sera from 13 patients were obtained repeatedly during dopaminergic anti-PRL treatment, to relate PRL level or anti-PRL treatment with the autoantibody levels. Elevated micro and/or macrovascular AECA were observed in sera from 19/25 patients (76%). Elevated titers of anti-CL Abs, all beta2-GPI-dependent, and low levels of anti-dsDNA antibodies (Abs) were also observed in the HPRL patients. Inhibition studies showed that the affinity purified AECAs bound the endothelial cell (EC) antigens in a dose-dependent manner. Titers of AECA as well as anti-DNA and anti-CL autoantibodies did not correlate with PRL level nor with the use or duration of anti-PRL treatment. None of the HPRL patients presented clinical manifestations of autoimmune disease. We conclude that elevated levels of AECA as well as anti-DNA and anti-CL autoantibodies are frequent in hyperprolactinemia. Our results further support the association of PRL and autoimmunity, and may point to a relationship between AECA-associated diseases and HRPL. The presence of autoantibodies in patients with HPRL might portend an increased risk for future development of autoimmune disease.