The interactions between bovine mononuclear cells and Haemophilus somnus are known to be complex. To study this interaction, a flow cytometric assay was developed to assess the effect of H. somnus on phagocytosis of killed opsonized Staphylococcus aureus by bovine alveolar macrophages and blood monocytes. Using this in vitro system, it was found that log phase H. somnus significantly inhibited the phagocytosis of killed opsonized S. aureus by bovine alveolar macrophages obtained both from healthy calves and from cattle experimentally infected with H. somnus. However, killed log-phase H. somnus, in vitro passaged and stationary phase H. somnus had no effect on the phagocytic activity of these cells. In contrast to bovine alveolar macrophages, blood monocytes showed a significant increase in their phagocytic activity following in vitro exposure to either log or stationary phase H. somnus. Using a lypophilic, non-toxic fluorophore PKH2 to label live H. somnus, it was possible to simultaneously measure the uptake of both S. aureus and H. somnus. Stationary and log phase H. somnus were taken up by macrophages equally well, even though phagocytosis of S. aureus was inhibited by only log phase H. somnus. These results demonstrate the ability of H. somnus to modulate bovine mononuclear phagocytic function which might contribute towards the pathogenesis of bovine hemophilosis.