Background Phenotyping sputum-resident leukocytes and evaluating their functional status are essential analyses for exploring the cellular basis of pathological processes in the lungs, and flow cytometry is widely recognized as the gold-standard technique to address them. However, sputum-resident leukocytes are found in respiratory samples which need to be liquefied prior to cytometric analysis. Traditional liquefying procedures involve the use of a reducing agent such as dithiothreitol (DTT) in temperature-controlled conditions, which does not homogenize respiratory samples efficiently and impairs cell viability and functionality. Methods Here we propose an enzymatic method that rapidly liquefies samples by means of generating O.sub.2 bubbles with endogenous catalase. Sputum specimens from patients with suspected pulmonary infection were treated with DTT, the enzymatic method or PBS. We used turbidimetry to compare the liquefaction degree and cell counts were determined using a hemocytometer. Finally, we conducted a comparative flow cytometry study for evaluating frequencies of sputum-resident neutrophils, eosinophils and lymphocytes and their activation status after liquefaction. Results Enzymatically treated samples were better liquefied than those treated with DTT or PBS, which resulted in a more accurate cytometric analysis. Frequencies of all cell subsets analyzed within liquefied samples were comparable between liquefaction methods. However, the gentle cell handling rendered by the enzymatic method improves cell viability and retains in vivo functional characteristics of sputum-resident leukocytes (with regard to HLA-DR, CD63 and CD11b expression). Conclusion In conclusion, the proposed enzymatic liquefaction method improves the cytometric analysis of respiratory samples and leaves the cells widely untouched for properly addressing functional analysis of lung leukocytes. Keywords: Cytometry of sputum immune cells, Catalase, Enzymatic liquefaction of respiratory samples, Hydrogen peroxide, Dithiothreitol