The process of neutrophilic polymorphonuclear leukocyte (PMN) migration in the bovine mammary gland during experimental Staphylococcus aureus challenge was studied using light and electron microscopies. The PMN were observed in various stages of margination in capillary lumens and migration into perivascular spaces. In infected tissue showing little evidence of epithelial damage, numerous PMN were accumulated in the perivascular space and aligned against the epithelial basal lamina, but not in the alveolar lumen. Other areas of infected tissue showed progressive tissue degeneration (autophagic vacuoles, vesiculation of endoplasmic reticulum, and cellular debris in the luminal milk) with the appearance of numerous PMN in lumens. The PMN were observed between the basal end of adjacent epithelial cells having intact tight junctions, but were never seen traversing the junctional complex. Some PMN lying between intact epithelial cells appeared to have ingested fat globules before entering the lumen. Although the mechanism by which PMN crossed tight junctions was not explained, alveoli with a portion of the epithelium sloughed off were frequently seen. The PMN appeared to be entering alveoli in large numbers at these damaged sites. There was also marked PMN infiltration in tissue from teat and gland cisterns of S aureus-infected quarters.