The persistent and viscous nature of airway secretions in cystic fibrosis disease leads to airway obstruction, opportunistic infection, and deterioration of lung function. Thioredoxin (Trx) is a protein disulfide reductase that catalyzes numerous thioldependent cellular reductive processes. To determine whether Trx can alter the rheological properties of mucus, sputum obtained from CF patients was treated with TRX and its reducing system [0.1 µM Thioredoxin reductase (TR) + 2 mM NADPH] and liquid phase:gel phase ratio (percent liquid phase) assessed by compaction assay. Exposure to low Trx concentrations (1 µM) caused significant increases in the percentage of liquid phase of sputum. Maximal increases in percent liquid phase occurred with 30 µM Trx. Additional measurements revealed that sputum liquefaction by the Trx reducing system is dependent on NADPH concentration. The relative potency of the Trx reducing system also was compared with other disulfide reducing agents. In contrast with Trx, glutathione and N-acetylcysteine were ineffective in liquefying sputum when used at concentrations below 1 mM. Sputum viscoelasticity, measured by magnetic microrheometry, also was diminished significantly following 20 minute treatment with 3, 10, or 30 µM Trx. Similarly, this reduction in viscoelasticty also was dependent upon NADPH concentration. Further investigation has indicated that Trx treatment increases the solubility of high molecular weight glycoproteins, and causes redistribution of extracellular DNA into the liquid phase of sputum. Recognizing that mucins are the major gel forming glycoproteins in mucus, we suggest that Trx alters sputum rheology by enzymatic reduction of glycoprotein polymers present in sputum.