A total of 457 serum samples from cattle kept under moderate tsetse challenge in Mukono County, Uganda, (79 of them with confirmed trypanosomosis) and 86 sera from cattle in Germany were tested for Trypanosoma antibodies using enzyme-linked immunosorbent assay (ELISA) with antigen obtained from blood stream form (BSF) and in vitro cultivated procyclic (PRO) trypanosomes. The BSF and PRO ELISA showed a moderate quantitative correlation. A difference plot revealed a slightly non-linear relation between the two methods. Cut-off values were established using (A) non-exposed controls, (B) exposed negative controls and (C) by mixture distribution analysis of the quantitative ELISA results for the sample from the endemic target population. The diagnostic agreement between both assays was significant (kappa, p < 0.05). The diagnostic accuracy of the two tests relative to standard parasitological detection methods was not markedly different as shown by the areas under the receiver operating characteristic (ROC) plots. In our study, neither non-exposed controls (cut-off A) nor exposed negative controls (cut-off B) were suitable for establishing cut-off values. Based on the cut-off value C, derived from mixture distribution analysis, the proportion of animals with elevated antibody levels in the study population was estimated by both assays at 45% (41-50% binomial 95% confidence interval). This can be regarded as an unbiased estimate of the proportion of 'sero-responders' and not necessarily as a proxy for infection prevalence in the target population. We recommend the PRO ELISA to be used for seroepidemiological surveys, since in vitro cultivation of procyclic trypanosomes allows a continuous and standardised preparation of test antigen.