This study evaluated the superantigen gene profiles, genetic relatedness and biological activity of exosecretions of 50 Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis. Genomic relatedness of S. aureus was determined by pulsed field gel electrophoresis analysis of macro-restricted chromosomes. The presence of genes encoding superantigens was confirmed by multiplex PCR. To study the biological activity of S. aureus exosecretions, the supernatants from bacterial liquid cultures were classified into three groups: those with leukotoxin-like properties, those with superantigen-like properties and those with no particular activity on leukocytes cultured in vitro. It was shown that all analyzed bacterial isolates belonged to the same clonal type and harbored the same combination of superantigen genes, namely sed, selj and ser. However, 22% of all isolates produced factors with superantigen-like and 48% of them with leukotoxin-like activities. Finally, although there were no detectable genetic differences between the analyzed bacterial isolates, the virulence factors secreted by them differed considerably.