N-oleoyl-heparin derivatives differing in their oleic acid and sulfate contents were synthesized and studied for their abilities to inhibit human leukocyte elastase (HLE), human leukocyte cathepsin G (CatG) and porcine pancreatic elastase (PPE) at pH 8.0, ionic strength 0.05 M and 37 degrees. Heparin (Hep) as well as N-oleoyl-heparins behaved as tight-binding, hyperbolic noncompetitive inhibitors of HLE (KiHep = 75 pM) and CatG (KiHep < 25 pM). The main driving force for the interaction between enzymes and glycosaminoglycans was electrostatic in nature. Under the condition [enzyme] >> Ki, the stoichiometries of the interaction with Hep were 1:2 (Hep:HLE) and 1:4 (Hep:CatG). Coupling one oleic acid residue to three disaccharide units of partially N-desulfated Hep, Ol1:3Hep, lowered HLE inhibition (Ki = 0.3 nM) and the stoichiometry of binding was reduced to 1:1. Re-N-sulfation of a similar derivative, Ol1:5Hep(SO4), containing one fatty acid residue for five disaccharide units, led to a substance with similar HLE inhibitory characteristics as Hep (Ki = 92 pM) and stoichiometry 1:2. Ol1:5Hep(SO4) was also a more efficient inhibitor of CatG (Ki < 33 pM) than Ol1:3Hep (Ki = 9.5 nM). The residual activities of N-oleoyl-Hep complexes with CatG were much lower than the corresponding activities in the presence of Hep. While oleate and Hep could not inhibit PPE, N-oleoyl-Hep, independently of fatty acid substitution and sulfate content, could inhibit this enzyme with Ki congruent to 60 nM and low residual activity. The efficient endopeptidase inhibitory characteristics of N-oleoyl-Hep derivatives, together with their non-anticoagulant properties and their capacity to interact with elastin, may be therapeutically useful in connective tissue degenerative diseases.