The definitive version is available at www3.interscience.wiley.com ; OBJECTIVE: The detection of trypanosome-specific antibodies in saliva is technically feasible, and, if clinically validated, could become an attractive option for non-invasive diagnosis of sleeping sickness. We wanted to optimize the test format of an enzyme-linked immunosorbent assay (ELISA)-based antibody detection system. METHODS: Different ELISA formats for antibody detection in serum and saliva were developed and standardized. Saliva and serum samples were collected from 78 patient and 128 endemic control samples, and sensitivity and specificity of saliva ELISAs, serum ELISAs and the card agglutination test for trypanosomiasis (CATT), were evaluated. RESULTS: All ELISA formats showed sensitivity and specificity above 90%. Saliva ELISAs showed a similar test performance as serum ELISAs and the CATT on whole blood or serum. CONCLUSIONS: This study confirms the potential of trypanosome-specific antibody detection in saliva.