The kinetics of the production of interferon gamma (IFN-gamma) in whole blood culture and its comparison with anti-listeriolysin O (ALLO) detection by ELISA were studied during oral infection of calves with Listeria monocytogenes. Culture filtrate antigen (CFA), listeriolysin O (LLO), and sonicated antigen (SA) were used to prime the peripheral blood mononuclear cells (PBMCs) and the plasma from orally infected calves. IFN-gamma and ALLO appeared as early as day 7 of an oral infection. IFN-gamma was detected earlier with LLO than with SA. The Max50 interleukin (IL-2) activity and IFN-gamma estimated in the culture supernatant from PBMCs primed in vitro with different antigens of L. monocytogenes revealed high induction of IL-2 and IFN-gamma by CFA, LLO and live antigen. IFN-gamma assay and ALLO detection were used for testing cases of repeat breeding in dairy cattle. It appeared that detection of IFN-gamma employing LLO can be used to diagnose listerial infections.