Protein C inhibitor (PCI) is a plasma serine proteinase inhibitor (serpin) that is a major physiological regulator of activated protein C. Inhibition of its target proteinase is accelerated by heparin in a reaction that involves the binding of both inhibitor and proteinase to heparin to form a ternary complex. This study was undertaken to understand the role of the H helix region (residues 264-278) of PCI in heparin binding and used (i) a recombinant truncated PCI fusion protein of the first 294 residues, (ii) H helix synthetic peptides containing single Arg/Lys-->Glu substitutions, and (iii) site-directed Ala mutagenesis of 4 basic residues (Arg-269, Lys-270, Lys-276, and Lys-277) in the H helix region of full-length recombinant PCI (rPCI) expressed in Baculovirus. The PCI fusion protein interfered in heparin-accelerated PCI-proteinase inhibition reactions, and it bound to heparin-Sepharose. Compared to the wild-type PCI fusion protein, deletion of the H helix from the fusion protein resulted in a reduction of both heparin-Sepharose binding and the ability to compete for heparin during PCI-proteinase inhibition reactions. Competition assays with H helix synthetic peptides revealed that the R269E altered peptide was the least effective at blocking heparin-catalyzed PCI-proteinase inhibition reactions. Compared with full-length active wild-type rPCI, R269A: K270A and K276A:K277A rPCI both had reduced heparin-Sepharose binding, but only R269A:K270A rPCI showed a loss of heparin-accelerated proteinase inhibition for both activated protein C and thrombin. We conclude that a major heparin-binding site of PCI is the H helix, unlike its heparin-binding serpin homologues antithrombin and heparin cofactor II, which bind heparin primarily through the D helix.