In cystic fibrosis (CF), actin and DNA originating from inflammatory cells contribute to the thickness of airway secretions. Actin can bind to DNA-rich fibers and potently inhibit the enzymatic activity of rhDNase. The in vitro effects of the actin-resistant rhDNase variant (A114R) were analyzed and compared with those of the wild-type rhDNase. Frozen and thawed CF airway secretions were incubated for 30 min with different concentrations (0.1, 0.5, 1, 5, or 10 microg/ml) of either actin-resistant rhDNase or wild-type rhDNase. We observed that both the wild-type and the actin-resistant rhDNase significantly decreased (p < 0.05 and p < 0.001, respectively) the airway secretion viscosity. The decrease in airway secretion viscosity was significant even at low concentrations (0.1 microg/ml) of the actin-resistant variant. Incubation with the actin-resistant variant resulted in a significant decrease (p < 0.02) of the airway secretion contact angle and cough transport. A significantly higher (p < 0.01) increase in contact angle and cough transport of airway secretions was observed at 10 microg/ml with the actin-resistant variant as compared with the wild-type rhDNase. The present study had demonstrated that the actin-resistant rhDNase variant (A114R) has an enhanced capacity to improve the physical properties and cough transport of airway secretions from patients with cystic fibrosis.