Previous studies have shown that exposure of human surfactant protein A (SP-A) to nitrating agents [peroxynitrite (ONOO); tetranitromethane (TNM; pH 8)] leads to nitrotyrosine formation. However, specific sites of nitration have not been identified. Herein, human SP-A, dissolved in Hepes buffer, was incubated with two boluses each of 0.5 mM ONOO- (pH 7.4) or 0.5 mM TNM (pH 8.0) for 15 min. After 30 min, SP-A samples were reduced, alkylated, and trypsin digested. The nitrated peptides and sites of amino acid nitration on the protein were identified by capillary high-performance liquid chromatography-coupled electrospray ionization tandem mass spectrometry (LC- ESMS/MS). The major nitrated peptide on both TNM- and ONOOexposed SP-A was the tryptic fragment Tyr161-Arg179 (YNTYAYVGLTEGPSPGDFR), located in the SP-A carbohydrate recognition domain. Sequencing of this nitrated peptide by LC-ESMS/MS demonstrated that the nitration was equally distributed on Tyr164 and Tyr166. A second lesser nitrated peptide corresponding to tryptic fragment Asn217-Arg222 (NCLYSR) was also found on TNM- and ONOOmodified SP-A. No other nitrated amino acid was detected. Nitrated SP-A exhibited decreased ability to aggregate surfactant lipids in the presence of Ca2+. These data demonstrate that nitration of a specific tyrosine decreased an important protein function.