Updated project metadata. The adhesion molecule CD99 is essential for transendothelial migration (TEM) of leukocytes. Here we demonstrate by biochemical and cellular assays that CD99 undergoes ectodomain shedding by the metalloprotease meprin β and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species.