Hemolysin was prepared from the culture supernatant of Haemophilus pleuropneumoniae, and its cytocidal and anti-phagocytic effects were investigated with pulmonary macrophages (PMP) obtained from the pig, mouse, guinea pig, or cotton rat. The hemolysin demonstrated both cytocidal and anti-phagocytic activities with the cotton rat, guinea pig, and pig PMP cells, but not with the mouse PMP cells. The species specificity in the cytocidal and anti-phagocytic effects with the PMP cells may be important in analyzing for the pathogenesis of the organisms to various animal species.