Macrophages by virtue of their phagocytic and antibacterial activities play an important role in the host resistance to intracellular pathogens including Listeria monocytogenes. However, the precise killing mechanism adopted by macrophages in the case of L. monocytogenes and the role of macrophage activation in bacterial killing are still unclear. In the present investigation, different antigens of pathogenic L. monocytogenes and three non-specific activators, namely, Lipopoly-saccharide (LPS), Phorbol Myristate Acetate (PMA) and Concanavalin A (ConA) supernatant were studied to adjudge their efficacy with regard to in vitro activation of bovine monocytes in terms of the production of reactive nitrogen intermediates (RNI), reactive oxygen intermediates (ROI) and their phagocytic index (PI). Of all the five antigens of L. monocytogenes, namely, viable bacteria used as live antigen (LA), heat-killed antigen (HKA), sonicated antigen (SA), culture filtrate antigen (CFA) and listeriolysin-O (LLO), LA turned out to be the best activator of monocytes for RNI as well as ROI production. In the PI assay, of the three antigens, that is, CFA, SA and LLO, CFA was found to be the best activator of phagocytosis followed by LLO and SA. Among non-specific activators, PMA induced the highest H2O2 production whereas LPS caused the maximum increase in RNI and PI production. On activation by different antigens of L. monocytogenes, the peripheral blood mononuclear cells (PBMCs) from infected animals produced significantly higher RNI than those from non-infected animals indicating the involvement of immune T-cells.