In this study, we isolated and characterized significantly purified CCSP-expressing cell populations from mouse lung by using high concentrations of collagenase and a flow cytometric sorting method. In addition, we showed that CCSP + cells expressed stem cell markers and form three dimensional spheroid colonies in culture. Furthermore, we confirmed that CCSP + cells may also express stemness characteristic in vivo as evidenced by label retention, the presence of CCSP/pro-SPC double positive BASCs and expression of stem cell markers in the epithelial lining of TBs of mice. Accordingly, the novel method described herein is a significant step in the progress of isolating and characterizing highly purified Clara cells in primary cultures.