Exosomes are small vesicles secreted by different immune cells and which display anti-tumoral properties. Stimulation of RBL-2H3 cells with ionomycin triggered phospholipase D2 (PLD2) translocation from plasma membrane to intracellular compartments and the release of exosomes. Although exosomes carry the two isoforms of PLD, PLD2 was enriched and specifically sorted on exosomes when overexpressed in cells. PLD activity present on exosomes was clearly increased following PLD2 overexpression. PLD2 activity in cells was correlated to the amount of exosome released, as measured by FACS. Therefore, the present work indicates that exosomes can vehicle signaling enzymes.