Maspin is a member of the serpin family with a reactive center loop that is incompatible with proteinase inhibition by the serpin conformational change mechanism. Despite this there are reports that maspin might regulate uPA-dependent processes in vivo. Using exogenous and endogenous fluorescence, we demonstrate here that maspin can bind uPA and tPA in both single-chain and double-chain forms, with K(d) values between 300 and 600 nM. Binding is at an exosite on maspin close to, but outside of, the reactive center loop and is therefore insensitive to mutation of Arg(340) within the reactive center loop. The binding site on tPA does not involve the proteinase active site, with the result that maspin can bind to S195A tPA that is already complexed to plasminogen activator inhibitor-1. The ability of maspin to bind these proteinases without involvement of the reactive center loop leaves the latter free to engage in additional, as yet unidentified, maspin-protein interactions that may serve to regulate the properties of the exosite-bound proteinase. This may help to reconcile apparently conflicting studies that demonstrate the importance of the reactive center loop in certain maspin functions, despite the inability of maspin to directly inhibit tPA or uPA catalytic activity in in vitro assays through engagement between its reactive center loop and the active site of the proteinase.