We have shown that low molecular weight dextran, as a potential mucolytic agent, reduced the viscoelasticity and spinnability of cystic fibrosis (CF) sputum and improved its ciliary transportability in vitro; it also reduced viscoelasticity of healthy dog mucus in in vitro testing. In anesthetized dogs, dextran administered by aerosol at 65 mg/mL increased tracheal mucus velocity, but this increase was not sustained for higher concentrations. The purpose of the present study is to evaluate whether low mol. wt. dextran sulfate, a charged oligosaccharide, exhibits similar effects to previously tested neutral dextran when administered by aerosol to anesthetized dogs in terms of mucus rheology and mucociliary clearance rate. Healthy mongrel dogs were anesthetized with pentobarbital and intubated. Aerosols of Ringer's solution or dextran sulfate (m.w. 5000) dissolved in Ringer's were generated by Pari LC STAR nebulizer, and delivered during 30-min periods of spontaneous breathing. Tracheal transepithelial potential difference (PD, using agar filled electrodes) and tracheal mucociliary velocity (TMV, by charcoal marker particle transport) were measured under bronchoscopic control, and mucus for viscoelasticity analysis by magnetic rheometry was collected by the endotracheal tube method. We performed experiments in seven dogs, involving 30-min administrations of aerosol, separated by 30-min periods of no aerosol. All dogs received inhalations of 6.5 mg/mL, 20 mg/mL, and 65 mg/mL dextran sulfate. Tracheal mucus viscoelasticity (average log G* over 1-100 rad/s) decreased progressively with increasing dose of dextran sulfate; for the highest concentration (65 mg/mL), log G* decreased by a factor of 2.61 (p = 0.021). A modest increase in the TMV was observed for the first dose of dextran sulfate (128% of baseline at 6.5 mg/mL, p = 0.066); thereafter TMV was stable. PD increased significantly at each concentration of dextran sulfate compared with Ringer control; however, there was no additional change between the three groups. The solids content of collected airway fluid (%SC) was gradually increased during successive 30-min dextran sulfate aerosols, indicating a significant residence time for the dextran in the mucus, and correlating with the decrease in viscoelasticity. These results suggest that dextran sulfate may be potentially of therapeutic value as a mucolytic agent, assisting mucus clearance by cough and physiotherapy, although whether it stimulates mucociliary clearance remains to be proven.