We analyzed the local antibody production in vitreous humor samples collected during vitrectomy in patients with severe vision-threatening uveitis. In 24 patients, paired serum and undiluted vitreous humor samples were collected and tested for antibodies against Toxoplasma gondii, herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, and Toxocara canis. Total IgG and the Goldmann-Witmer coefficient were determined. The initial diagnosis of ocular toxoplasmosis could be confirmed in six of the seven patients. The seventh patient showed a local antibody production against herpes simplex virus. One of the three patients with chronic panuveitis at initial diagnosis showed a local antibody production against T. gondii. These last two findings resulted in a change in medical treatment. Analysis of local antibody production in vitreous humor samples is a valuable diagnostic tool.