The main purpose of this work was to study the generation of Angiotensin I-converting enzyme inhibitory (ACEI) peptides after gastrointestinal digestion of pork meat by the action of pepsin and pancreatin at simulated gut conditions. The hydrolysate was further subjected to reverse phase chromatography in order to separate the fractions with ACEI activity. Using MALDI-TOF/TOF mass spectrometry, 12 peptides were identified in these fractions. It is worth highlighting the novel peptides ER, KLP, and RPR with IC(50) values of 667 microM, 500 microM, and 382 microM, respectively. Results obtained by MALDI-TOF/TOF mass spectrometry were complemented by a second approach consisting of the analysis of the hydrolysate directly by nanoLC-ESI-MS/MS followed by a study of the obtained sequences and comparison with known ACEI peptide sequences. By using these two approaches, a total of 22 peptides were selected for its synthesis and further in vitro assay of ACEI activity. The strongest ACE inhibition was observed for peptide KAPVA (IC(50) = 46.56 microM) followed by the sequence PTPVP (IC(50) = 256.41 microM). Sequence similarity searches revealed that these two peptides derive from muscle titin, constituting the first identified ACEI peptides coming from this protein. This is also the first time that ACEI sequences MYPGIA and VIPEL have been reported. Other identified and synthesized sequences showed less ACEI activity. The obtained results evidence the potential of pork meat proteins as a source of antihypertensive peptides after gastrointestinal digestion.