The cellular prion protein (PrP(c)) is physiologically cleaved in the middle of its 106-126 amino acid neurotoxic region at the 110/111 downward arrow112 peptidyl bond, yielding an N-terminal fragment referred to as N1. We recently demonstrated that two disintegrins, namely ADAM10 and ADAM17 (TACE, tumor necrosis factor alpha converting enzyme) participated in both constitutive and protein kinase C-regulated generation of N1, respectively. These proteolytic events were strikingly reminiscent of those involved in the so-called "alpha-secretase pathway" that leads to the production of secreted sAPPalpha from betaAPP. We show here, by transient and stable transfection analyses, that ADAM9 also participates in the constitutive secretion of N1 in HEK293 cells, TSM1 neurons, and mouse fibroblasts. Decreasing endogenous ADAM9 expression by an antisense approach drastically reduces both N1 and sAPPalpha recoveries. However, we establish that ADAM9 was unable to increase N1 and sAPPalpha productions after transient transfection in fibroblasts depleted of ADAM10. Accordingly, ADAM9 is unable to cleave a fluorimetric substrate of membrane-bound alpha-secretase activity in ADAM10(-/-) fibroblasts. However, we establish that co-expression of ADAM9 and ADAM10 in ADAM10-deficient fibroblasts leads to enhanced membrane-bound and released fluorimetric substrate hydrolyzing activity when compared with that observed after ADAM10 cDNA transfection alone in ADAM10(-/-) cells. Interestingly, we demonstrate that shedded ADAM10 displays the ability to cleave endogenous PrP(c) in fibroblasts. Altogether, these data provide evidence that ADAM9 is an important regulator of the physiological processing of PrP(c) and betaAPP but that this enzyme acts indirectly, likely by contributing to the shedding of ADAM10. ADAM9 could therefore represent, besides ADAM10, another potential therapeutic target to enhance the breakdown of the 106-126 and Abeta toxic domains of the prion and betaAPP proteins.