Tobramycin, an aminoglycoside antibiotic, is used in the treatment of Pseudomonas aeruginosa infections in cystic fibrosis patients. Tobramycin bioactivity, however, is antagonized by sputum. Glycoproteins (mucins) and high-molecular-weight DNA make up 2 to 3% (P. L. Masson and J. F. Heremans, p. 412-475, In M. J. Dulfano, ed., Sputum: Fundamentals and Clinical Pathology, 1973) and 3 to 10% (W. S. Chernick and G. J. Barbero, Pediatrics 24:739-745, 1959, and R. Picot, I. Das, and L. Reid, Thorax 33:235-242, 1978) of the dry weight of sputum, respectively. tobramycin binds to both mucins and DNA obtained from sputum (R. Ramphal, M. Lhermitte, M. Filliat, and P. Roussel, J. Antimicrob. Chemother. 22:483-490, 1988). In vitro, recombinant human DNase (rhDNase) hydrolyzes high-molecular-weight DNA of > 50 kb within sputum to fragments of 2 to 4 kb. Studying dialyzable tobramycin, we examined drug binding to whole sputum and to "mock sputum," which consisted of porcine gastric mucin and calf thymus DNA. We also studied the effects of rhDNase treatments of sputum, mock sputum, and calf thymus DNA on tobramycin binding. We found that treatments of sputum, mock sputum, and calf thymus DNA with rhDNase did not significantly increase the tobramycin bioactivity within the dialysates; surprisingly, sputum binding of tobramycin was increased by rhDNase. We conclude that rhDNase does not increase the bioactivity of tobramycin in sputum.