Cystic fibrosis (CF) patients frequently experience recurring airway infections characterized by thick, viscous sputum. The consistency and nature of these purulent secretions may produce a significant barrier to the successful delivery of drugs and gene therapy vectors designed to treat CF. We have carried out a series of in vitro studies to determine the distribution of two macromolecular components typically present in purulent sputum, bacterial alginate and neutrophil-derived DNA. Sputum samples were obtained from hospitalized CF patients. DNA and alginate were disrupted, respectively, by the in vitro additions of human recombinant deoxyribonuclease I (rhDNase) or alginate lyase prepared from a mucoid strain of Pseudomonas aeruginosa. N-acetyl-L-cysteine (acetylcysteine) was similarly used to collapse the mucin matrix of these samples for comparison. Using a centrifugation-based rheological method known as the compaction assay, a greater maximal response was observed for rhDNase compared to alginate lyase treatment. A simultaneous addition of these enzymes to purulent sputum produced an additive compaction response. Electron microscopy was used to identify alginate and DNA components within the mucin matrix of sputa and to evaluate changes following treatment with high concentrations of alginate lyase or rhDNase. DNA was more widely distributed throughout purulent samples than alginate. Differences in the distribution of DNA and alginate may explain, at least in part, the larger compaction response to rhDNase versus alginate lyase treatment. An improved understanding of DNA and alginate distribution within purulent CF sputum may lead to improvements in drug and vector delivery to airway epithelial cells.