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Background: Currently, the identification of Clara cell and type II alveolar epithelial cell-type cancer cells requires electron microscopy, which is a time-consuming and expensive process involving a complicated tissue sampling procedure. The aim of this study was to identify unique biomarkers for Clara cell and type II alveolar epithelial cell-type lung cancer cells, respectively, with proteomic profiling. Methods: Six human lung adenocarcinoma cell lines (A549, NCI-H358, NCI-H1650, HCC827, NCI-H1395, and NCI-H1975) were investigated for their ultrastructural characteristics. The differentially expressed proteins (DEPs) were screened between NCI-H358 cells (Clara cell type) and A549 cells (type II alveolar epithelial cell type) using two-dimensional difference gel electrophoresis (2D-DIGE) and matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS), and then they were validated by western blot. The protein expression levels of endoplasmic reticulum oxidoreductin 1-α (ERO1L), Clara cell 10-kD protein (CC10), and surfactant protein C (SP-C) were also determined in the six cell lines assayed. Results: NCI-H358 cells featured Clara cell differentiation; A549, NCI-H1975, and HCC827 cells had characteristics of type II alveolar epithelial cells; and NCI-H1395 and NCI-H1650 cells had no differentiation characteristics of any lung adenocarcinoma cell type. Five DEPs including ubiquitin carboxyl- terminal hydrolase isozyme L1 (UCHL1), cytokeratin 19 (CK19), cytokeratin 8 (CK8), ERO1L, and peroxiredoxin 2 (PRDX2) between NCI-H358 and A549 cells were identified for further validation; however, none of them showed suitability as an effective biomarker. Similarly, CC10 and SP-C were not appropriate biomarkers. Conclusions: Cytological subtypes of NCI-H1975 and HCC827 cells were identified, but no promising biomarker was discovered in the present study.