The long-term goal of our study is to identify chronic obstructive pulmonary disease (COPD)-related bronchoalveolar lavage fluid (BALF) nitroproteins to clarify COPD pathological mechanisms and to discover biomarkers of COPD. The goal of the present study was to detect the presence of, and potential roles of, nitroproteins in, human ex-smoker (without COPD) BALF samples. Nitroproteins were immunoprecipitated from two separate BALF samples, and digested with trypsin; and tryptic peptides were analyzed with matrix-assisted laser desorption/ionization (MALDI)-tandem mass spectrometry (MS/MS). Each MS/MS spectrum was composed of accumulated scans (n = 50-100). The MS/MS data were searched with BioWorks 2.0 TuboSequest in the SwissProt database to generate the amino acid sequence, which was evaluated manually. Eleven nitrotyrosine sites were identified in eight proteins, including progestin and adipoQ receptor family member III, zinc finger protein 432, proteasome subunit alpha type 2, NADH-ubiquinone oxidoreductase B14, slit homolog 1 protein, lysozyme, aldose 1-epimerase, and PTS system lactose-specific EIICB component. Each nitrotyrosine site was located within a specific protein domain and motif. Those identified nitrated proteins could be involved in multiple functional metabolic systems, including transcriptional regulation, mitochondrial complex, immune system, and energy metabolism.