In this study, differential display polymerase chain reaction (PCR) was used to search for unique or enhanced expression of genes in prevalent bovine mastitis-causing Staphylococcus aureus strains. Comparison of a pair of prevalent and rare strains revealed the differential expression of several genes. The lactose-specific permease, enzyme II (EII), was highly expressed in the prevalent strain. This gene was selected for further study due to its potential influence on bacterial growth, because lactose is the primary carbohydrate in milk. Growth analysis illustrated that prevalent strains reach significantly higher growth densities sooner than rare strains. Quantitative competitive reverse transcription PCR (QC RT-PCR) revealed increased EII mRNA expression in prevalent strains as compared to rare strains. Mutation of the EII gene resulted in abrogated growth and decreased EII mRNA expression in media containing lactose. These data suggest that increased EII expression may facilitate the pathogenesis of S. aureus mastitis by enhancing growth. This study is the first to implicate EII as a potential virulence factor in mastitis, and therefore may be useful in the development of novel therapeutic strategies against S. aureus mastitis.