Following activation of granulocytes, L-selectin (CD62L) is generally shed from the cellular surface, whereas Mac-1 (CD11b/CD18) expression is well known to increase. However, a number of studies in bovines and humans show that the expression of L-selectin may increase as well. This urged us to examine the possible existence of both L-selectin and Mac-1 reservoirs in bovine neutrophil and eosinophil populations through the use of flow cytometry in combination with an optimized method for cell membrane permeabilization. Augmented L-selectin and Mac-1 expression was detected in both granulocyte populations upon saponin treatment. Confocal microscopic studies indicated that both molecules exhibit a different pattern of subcellular localization. Incubation with sialidase revealed the existence of hidden L-selectin epitopes at the cell surface, while no additional Mac-1 epitopes were exposed. Platelet-activating factor stimulation decreased surface and total expression of L-selectin to the same extent in both populations, but solely affected Mac-1 surface expression on eosinophils. Moreover, cytoskeletal actin filaments and microtubules were found to be involved in the regulation of Mac-1 surface expression on bovine neutrophils and eosinophils. In marked contrast, expression of L-selectin was minimally affected by cytoskeleton perturbing agents. The present study indicates that L-selectin and Mac-1 adhesion molecules reside in distinctly located reservoirs in bovine granulocytes and can be selectively mobilized upon in vitro stimulation.