Thrombospondin 1 was recently shown to bind to and inhibit the activity of neutrophil elastase (Hogg, P. J., Owensby, D. A., Mosher, D. F., Misenheimer, T. M., and Chesterman, C. N. (1993) J. Biol. Chem. 268, 7139-7146). This finding led us to question whether thrombospondin 1 also binds and inhibits the other major serine proteinase of neutrophils, cathepsin G. In a competitive binding assay, cathepsin G bound to thrombospondin 1 reversibly and saturably with a dissociation constant in the low nanomolar range. The kinetic mechanism of inhibition of cathepsin G activity by thrombospondin 1 was determined using the synthetic cathepsin G substrate, Suc-Ala-Ala-Pro-Phe-p-nitroanilide, and is consistent with hyperbolic tight-binding inhibition in which thrombospondin 1 binds cathepsin G and the Michaelis cathepsin G-substrate complex and weakens, but does not abolish, the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide. In the presence of 2 mM calcium ions, 2.9 +/- 0.4 mol of cathepsin G interacted with 1 mol of thrombospondin 1 trimer with a site-binding constant of 7.0 +/- 3.5 nM, which reduced the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide 8.5 +/- 1.4-fold. A lower limit for the on rate constant of 5 x 10(6) M-1 S-1 was established. The affinity of binding and stoichiometry for the interaction between cathepsin G and thrombospondin 1 was enhanced in the absence of calcium ions. In the presence of EDTA, 5.3 +/- 0.5 mol of cathepsin G interacted with 1 mol of thrombospondin 1 with a site-binding constant of 2.1 +/- 1.6 nM, implying the existence of two binding sites for cathepsin G on each subunit of thrombospondin 1, one or both of which is variably exposed and sensitive to calcium ions. Thrombospondin 1 protected fibronectin from cleavage by cathepsin G and blocked cathepsin G-mediated platelet aggregation. In summary, the binding of cathepsin G to thrombospondin 1 is tight, reversible, and close enough to the active site of cathepsin G to perturb the interactions of a small synthetic substrate and exclude a macromolecular protein substrate and platelets. Using defined proteolytic fragments and different conformers of thrombospondin 1, the binding sites for cathepsin G have been localized to the thrombospondin 1 type 3 repeats.