The bovine mammary gland requires lymphocytes for immune protection of the gland from foreign pathogens and, in addition, to transfer immune protection to the neonate via colostrum and milk. The process of homing primed lymphocytes to tissues is mediated by the interaction of cell-adhesion molecules displayed on the surface of lymphocytes and counter receptors displayed on the vascular endothelium. This study was conducted to identify the cell-adhesion molecules involved in homing lymphocytes to the bovine mammary gland at four different physiological stages; pregnant, colostral, lactation and involution. The expression and distribution of adhesion molecules in alveolar tissues and supramammary lymph nodes from the mammary glands of healthy cows was determined in situ by immunohistochemical analysis and compared with bovine Peyer's patch, used as a typical mucosal-associated lymphoid tissue and positive control. The mucosal addressin molecule, MAdCAM-1, was not detected in bovine mammary tissues at any of the four different physiological stages. Absence of MAdCAM-1 expression was verified by quantitative real-time RT-PCR analysis. Transcription levels of MAdCAM-1 mRNA were found to be more then 5 × 103-fold lower in mammary alveolar tissues compared with bovine Peyer's patch tissues. In contrast to MAdCAM-1, phase-dependent protein expression of VCAM-1 was detected in both mammary alveolar tissues and the supramammary lymph nodes, with the highest expression observed in colostral phase cows. The protein expression in mammary alveolar tissues was limited to larger venules, although in colostral phase cows, VCAM-1 was also detected around the alveoli perimeter. In the supramammary lymph node, VCAM-1 protein was observed on both small and large venules. PNAd was detected in supramammary lymph nodes at all physiological stages of the mammary gland; however, it was not found in mammary alveolar tissues. Lymphocytes expressing β7 were not detected in mammary tissues and lymphocytes expressing CD62L were only observed in the supramammary lymph nodes. Overall the data suggest that MAdCAM-1 and VCAM-1 are not involved in homing lymphocytes to the bovine mammary gland; whereas, VCAM-1 and PNAd may have this role in the supramammary lymph node.