Heparin cofactor II (HCII) is plasma glycoprotein and thrombin inhibitor of the serpin type previously shown to inhibit thrombin in the absence of its N-terminal 74 amino acids, and to be cleaved by neutrophil elastase (NE) at two sites: I66-F67 and V439-G440, the P6-P5 bond of the reactive center loop. We examined the contribution of Val439 to the reaction of HCII with thrombin and NE. Hexahistidine-tagged HCII proteins lacking residues 1-66 (H6Δ66HCII) containing either the wild-type Val 439 or one of six substitutions were expressed in E. coli. The rates of heparin-catalyzed thrombin inhibition of the V439L, C, or R variants were reduced at least 80-fold compared to wild-type H6Δ66HCII, while those of the F, S, or W variants were largely unchanged. Following controlled exposure to NE in the presence of heparin, these latter variants retained 3.5to 4.5-fold more residual anti-thrombin activity than wild-type H6Δ66HCII treated in the same manner. This resistance arose due to deflection of NE attack from V439-G440 to secondary sites. The F, S, or W V439 variants exhibited a similar or greater degree of NE resistance when re-expressed as full-length hexahistidine-tagged HCII proteins, suggesting that the I66-F67 NE site is not well recognized in non-glycosylated HCII. Of these full-length variants, the V439F was the most active, exhibiting only a 2-fold reduction in its heparin-catalyzed rate of thrombin inhibition. HCII can therefore be made NE-resistant without severely compromising its capacity to inhibit thrombin.