To see whether detection of antibodies against listeriolysin O (LLO) could be used to diagnose human listeriosis, sera from 28 patients infected with Listeria monocytogenes and 101 controls were tested by dot-blot titration with purified LLO. 27 patients (96.4%) with listeriosis produced specific anti-LLO. Anti-LLO was detected in 8 (15.6%) of 51 healthy controls and in 6 (12.0%) of 50 controls who had various bacterial, fungal, and viral infections. Anti-LLO titres did not exceed 100 in these two control groups. Anti-LLO could be detected soon after clinical onset of listeriosis, and antibodies persisted for at least several months. This test might be useful for epidemiological surveys and for serodiagnosis of listeriosis, especially when bacteria cannot be isolated.