Background: Flow cytometry (FCM) provides an alternative to radioactive methods for the analysis of T-cell responses. However, a comparative study of common FCM assays in an outbred ruminant model is lacking, which motivated this work. Methods: Goats immunized with the obligate intracellular bacterium Cowdria ruminanthium, inactivated and emulsified in oil-based adjuvants, were used as a model to study T-cell recall responses in vitro. FCM-based methods to measure Cowdria-induced lymphoblastogenesis, DNA synthesis, and interleukin-2 receptor (IL-2R) expression by T-cell subsets were compared. Results: IL-2R expression was the most sensitive and reliable method provided that the number of molecules per cell was analyzed and not simply the percentage of positive cells of a given phenotype. Despite high back-ground due to adjuvant and low proliferation, this method could detect antigen-specific activation of immune CD4+ and CD8+ T cells. Conclusions: FCM-based measurement of lymphoblastogenesis and DNA synthesis are not the most appropriate methods to analyze T-lymphocyte activation during vaccination of outbred animals. On several occasions, analysis of IL-2R expression was the only assay capable of discriminating between vaccinated and naive animals in this model.