Staphylococcus aureus is a frequent cause of mastitis in dairy cows. However, pathogenesis of the infection has not been completely defined. We report the invasion of two strains of S. aureus into a bovine mammary epithelial cell line and a bovine mammary epithelial cell primary culture. Invasion of S. aureus into bovine mammary cells was time-dependent. Transmission electron microscopy of bovine mammary cells invaded by S. aureus showed intracellular replication of the bacterium within membrane-bound vacuoles. Invasion was reduced significantly when bovine mammary epithelial cells were treated with inhibitors of F-actin microfilament polymerization but not when these cells were treated with inhibitors of microtubule formation. Results indicated that S. aureus is capable of invading and replicating inside bovine mammary epithelial cells. Data also suggested that S. aureus invasion of bovine mammary epithelial cells requires active participation of specific components of the cytoskeleton of the epithelial cell.