Research on mastitis in dairy cows caused by Escherichia coli has reported the emergence of strains capable of inducing chronic mastitis and that these strains adhered to and internalized into bovine mammary epithelial cells better than strains of E. coli isolated from acute mastitis. To understand mechanisms and strategies used by chronic E. coli strains to survive intracellularly internalization studies using bovine mammary epithelial cells treated with inhibitors of caveolae-mediated endocytosis (CME) and receptor-mediated endocytosis (RME), double immunofluorescence labeling confocal laser and fluorescence microscopy were conducted. Internalization studies showed that strains chronic E. coli strains persisted intracellularly longer than acute E. coli strains. Treatment of bovine mammary epithelial cells CME or RME inhibitors resulted in lower numbers of intracellular E. coli strains associated with chronic or acute mastitis than untreated controls. In addition, when selective CME inhibitors were used significantly fewer chronic E. coli were detected intracellularly than acute E. coli or untreated controls. Confocal laser microscopy showed that chronic E. coli strains colocalized preferentially with caveolae whereas acute strains did so with early endosomes, an early step of RME. These results suggest that strains of E. coli associated with chronic mastitis exploit lipid rafts/CME to internalize into and move through mammary epithelial cells. By exploiting this endocytosis pathway, chronic E. coli strains avoid bactericidal mechanisms such as endosome acidification and endosome-lysosome fusion, thus allowing intracellular survival. Data from this study helps to explain how these strains are capable of causing chronic E. coli mastitis.