Lung epithelial cells play critical roles in initiating and modulating immune responses during pulmonary infection or injury. To better understand the spectrum of immune response-related proteins present in lung epithelial cells, we developed an improved method of isolating highly pure primary murine alveolar type (AT) II cells and murine tracheal epithelial cells (mTECs) using negative selection for a variety of lineage markers and positive selection for epithelial cell adhesion molecule (EpCAM), a pan-epithelial cell marker. This method yielded 2-3 × 106 ATII cells/mouse lung and 1-2 × 104 mTECs/trachea that were highly pure (>98%) and viable (>98%). Using these preparations, we found that both ATII cells and mTECs expressed the Lyn tyrosine kinase, which is best studied as an inhibitory kinase in hematopoietic cells. However, we found little or no expression of Syk in either ATII cells or mTECs, which is in contrast to earlier published reports. Both cell types expressed C-type lectin receptors, anaphylatoxin receptors, and various Toll-like receptors (TLRs). In addition, stimulation of ATII cells with TLR ligands led to secretion of various cytokines and chemokines. Interestingly, lyn-/- ATII cells were hyperresponsive to TLR3 stimulation, suggesting that, as in hematopoietic cells, Lyn might be playing an inhibitory role in ATII cells. In conclusion, the improved isolation method reported here, along with expression profiles of various immune defense proteins, will help refocus investigations of immune-related signaling events in pulmonary epithelium.