Neutrophils are present in milk of cows as a means of suppressing invading pathogens during mastitis. However, the manner by which neutrophils traverse the secretory epithelia is still not clear: do they diapedese between epithelial cells or do they kill epithelial cells to gain entry into milk? We investigated the process of bovine neutrophil diapedesis across bovine mammary gland epithelium in vitro. The bovine mammary epithelial cell line MAC-T, grown on collagen-coated filters, formed a confluent monolayer with characteristic tight junctions, basal-apical polarity, and functional barriers to the dye trypan blue. Neutrophils added on the apical surface of the monolayer were stimulated to diapedese across the epithelium by the addition of Staphylococcus aureus (10(7) colony-forming units/ml) to the basal compartment. Light and transmission electron microscopy revealed the series of events for neutrophil transmigration: accumulation of neutrophils on the surface of epithelial monolayer; projection of pseudopods into intercellular junctions and movement of neutrophils between adjacent epithelial cells; and reapproximation of the lateral epithelial cell membranes and reformation of the apical tight junctions after neutrophils crossed the epithelium. Morphologically, epithelial cell damage caused by neutrophil diapedesis was not evident. This in vitro model provides a two-dimensional epithelial sheet by which neutrophil diapedesis can be qualitatively studied under defined conditions. Results of the study suggest a major mode by which bovine neutrophils diapedese across the alveolar epithelia into milk during mastitis.