Virulence of Legionella pneumophila strain Monza 3 and Staphylococcus aureus strain Smith diffuse was investigated after sequential cultures on nutrient media. L. pneumophila lost its ability to multiply within Acanthamoeba polyphaga after 50 passages on Legionella selective agar, while S. aureus maintained its pathogenicity in the mouse peritonitis model after 100 sequential cultures on tryptic soy agar. These results demonstrate high preservation of virulence in staphylococci in contrast to legionellae. Differentiation of virulent and avirulent phenotypes of legionellae by the protozoal model may be helpful for detecting sources of infection in water hygiene.