Gelatinase A, a member of the matrix metalloproteinase family, contains three fibronectin type II (FnII)-like modules that are inserted within its catalytic domain. These FnII modules, defined as exosites, play an essential role in targeting the enzyme to matrix macromolecules, a process which can down-regulate membrane-type metalloproteinase-driven progelatinase A activation. The exosite/substrate-directed gelatinase inhibitors has been proposed as an alternative approach to disappointing active site-directed inhibitors, to control gelatinase A activity. In preliminary experiments, we evidenced that long-chain unsaturated fatty acids could bind preferentially to the first FnII module of gelatinase A. This interaction inhibits the activity of this enzyme towards proteins (type I gelatin and collagen) and an octapeptide substrate, with K(i) in the micromolar range. Since gelatinase A-catalyzed matrix proteolysis might display a positive or negative influence (depending on the substrate cleaved), the design of exosite-specific compounds for noncatalytic targeting of gelatinase A would necessitate an extensive degradomic analysis.