Compelling evidence suggests that nitric oxide (NO*), a metabolite of arginine, plays an important role in wound healing. Arginine is a semi-essential amino acid that is metabolized by nitric oxide synthase and arginase. One model for wound-healing regulation suggests the importance of strict reciprocal control of these enzymes in wounds. The purpose of this pilot study was to investigate arginine metabolism in wound fluids from patients with Stage III or IV pressure ulcers receiving negative pressure wound therapy (NPWT). Wound fluids were collected from 8 patients, aged 31-79 years, before and after initiation of NPWT on Days 1, 3, and 7. Wound fluids were analyzed for nitrates/nitrites (NOx), arginine, citrulline, proline, and ornithine. There were no significant differences between NOx, arginine, citrulline, proline, and ornithine concentrations before and after initiation of NPWT among the various timepoints. However, we observed a downward trend of NO* levels from baseline to Day 7 of NPWT treatment. Furthermore, we detected a decrease in arginine levels over the study period, suggesting that the iNOS/citrulline pathway predominated during the first 72 hr of treatment, and the arginase/ ornithine pathway dominated thereafter. Arginine and its metabolites are detectable in wound fluids from patients with Stage III or IV pressure ulcers on NPWT. Further studies on chronic wounds are warranted to correlate wound-healing outcomes with arginine metabolites at the cellular and molecular level over a longer period of time.