Members of the ADAM (a disintegrin and metalloproteinase) family of proteins possess a multidomain architecture which permits functionalities as adhesion molecules, signalling intermediates and proteolytic enzymes. ADAM8 is found on immune cells and is induced by multiple pro-inflammatory stimuli suggesting a role in inflammation. Here we describe an activation mechanism for recombinant human ADAM8 that is independent from classical PC (pro-protein convertase)-mediated activation. N-terminal sequencing revealed that, unlike other ADAMs, ADAM8 undergoes pre-processing at Glu(158), which fractures the Pro (pro-segment)-domain before terminal activation takes place to remove the putative cysteine switch (Cys(167)). ADAM8 lacking the DIS (disintegrin) and/or CR (cysteine-rich) and EGF (epidermal growth factor) domains displayed impaired ability to complete this event. Thus pre-processing of the Pro-domain is co-ordinated by DIS and CR/EGF domains. Furthermore, by placing an EK (enterokinase) recognition motif between the Pro- and catalytic domains of multiple constructs, we were able to artificially remove the pro-segment prior to pre-processing. In the absence of pre-processing of the Pro-domain a marked decrease in specific activity was observed with the autoactivated enzyme, suggesting that the Pro-domain continued to associate and inhibit active enzyme. Thus, pre-processing of the Pro-domain of human ADAM8 is important for enzyme maturation by preventing re-association of the pro-segment with the catalytic domain. Given the observed necessity of DIS and CR/EGF for pre-processing, we conclude that these domains are crucial for the proper activation and maturation of human ADAM8.