Angiotensin converting enzyme (ACE, E.C. 3.4.15.1) is a metallopeptidase with two homologous domains, with a well-known activity on Renin-Angiotensin system and bradykinin hydrolysis. The enzyme also cleaves C-terminal dipeptides from substrates with free carboxyl groups with a wide specificity. It´s well-described ACE as a somatic (N and C domains), germinal (C-domain) forms, and an ileal fluid form containing only the N-domain. Ileal ACE form shows a molecular mass of 108 kDa and is less chloride dependent. An association between ileal ACE and cell differentiation is described also. The hydrolytic properties on several natural and synthetic substrates of somatic and germinal isoforms are well stablished. Meanwhile, the N-domain isoform substrate hydrolyses are unexplored. Several hormones affect the gastrointestinal motility as enteroglucagon and YY peptide. The aim of this study was characterize the ACE activity in human ileal fluid with internally quenched fluorescent peptides. The ileal fluid was obtained during surgical procedures and the purification outline included a dialysis of biological fluid against 0.1 M sodium phosphate, containing NaCl 0.05 M, pH 7.0, followed by a gel filtration step in a Superdex ä 200 10/300 column. Enzymatic activity was determined using the synthetic substrate AbzFRK(Dnp)-P-OH at pH 7.0 and Hip-His-Leu at pH 8.3. SDS-PAGE was used to determine the protein purification grade. Our results showed the partial isolation of two forms of ACE. The main form showed a molecular mass above 100 KDa that corresponded to a 2/3 of total ACE activity of ileal fluid. The other form showed a molecular mass similar to the somatic ACE form. The two substrates were hydrolyzed by the isolated enzymatic forms. Both activities were abolished by captopril. These data showed that ileal fluid contains ACE activity and this activity is devoid to the action of distinct ACE isoforms. If these enzymes have some role in hydrolysis of gastrointestinal peptides or act on mucosa secretion mechanisms is unknown. Finacial support: CNPq, FAPEMIG, UFTM.