Little is known about bacterial and host factors that contribute to the establishment and persistence of intramammary infection by Streptococcus dysgalactiae. Streptococcus dysgalactiae adheres to epithelial cells from the bovine mammary gland and to extracellular matrix proteins in vitro and invades mammary epithelial cell cultures, all of which can be potentially important pathogenic mechanisms. In this study, mechanisms involved in the invasion of Strep. dysgalactiae into epithelial cells from the bovine mammary gland were characterized. Studies were conducted to determine whether Strep. dysgalactiae invaded mammary epithelial cell cultures in a dose-dependent manner and whether mammary epithelial cells that harbored different numbers of Strep. dysgalactiae for varying times were damaged. Bacterial invasion increased as inoculum size increased; however, the number of intracellular bacteria was not proportional to the inoculum size, increased; however, the number of intracellular bacteria was not proportional to the inoculum size, indicating that a finite number of organisms is capable of invading epithelial cells. No net increase in intracellular organisms was detected at any bacterial density evaluated; however, Strep. dysgalactiae remained viable throughout the evaluation. In addition, Strep. dysgalactiae did not appear to cause cell injury at any bacterial density or time point evaluated. These data suggest that Strep. dysgalactiae can survive within mammary epithelial cells for an extended time without losing viability or damaging the eukaryotic cell. This feature may be associated with the development of persistent infection and protection of organisms from antimicrobial drugs and host defense mechanisms and may provide a route for bacterial colonization of subepithelial tissues.