The present work intended to initiate an unusual approach aiming at combining enzymatic and electron paramagnetic resonance (RPE) spectroscopy of paramagnetically labelled peptide substrates. Angiotensin-I-converting enzyme (ACE, EC 3.4.15.1), a dipeptidyl carboxypeptidase which the main function is to cleave the C-terminal dipeptide from angiotensin I (AI, DRVYIHPFHL) to produce the potent vasoconstrictor angiotensin II (AII) was selected for the present study. Two AI analogues containing the TOAC spin label (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) [1,2] at positions 0 and 9 were synthesized and both (TOAC⁰- and TOAC⁹-AI) were initially submitted to ACE catalytic experiments comparatively to the model AI substrate. The internally labeled peptide which contains the TOAC moiety at P`1 position of the ACE cleavage site (TOAC⁹-AI) was not a substrate regardless the amount of enzyme used in standardized protocol early described. Otherwise the N-terminally labeled TOAC⁰-AI was normally cleaved at the 8-9 peptide bond yielding His-Leu and the TOAC⁰- AII products. The catalytic efficiency for this paramagnetic substrate was determined through HPLC experiments where the amount of one of formed product (TOAC⁰-AII) was calculated through its peak area in the chromatograms. In close accordance with previous results, the model AI substrate gave K_m, k_cat and k_cat/K_m values of 34.6 mM, 500.7 min^-1 and 14.6 mM^-1.min^-1, respectively. Comparatively, no significant difference was detected for the TOAC⁰-AI analogue which yielded 11.56 mM, 191.0 min^-1 and 16.5 mM^-1.min^-1, respectively. The following step involved the evaluation of secondary structure information regarding these TOAC-analogues comparatively to AI and AII. The CD experiments varying the pH of the medium or the amount of secondary structure inducing TFE depicted a clear conformational differentiation between both paramagnetic analogues. Initial conformation studies through EPR approach indicated that TOAC⁰-AI and TOAC⁹-AI analogues present rotational correlation time (t) values of about 2 x 10^-10 s and 5 x 10^-10 s, respectively. Other experiments including the synthesis of additional analogues have been already designed to deeper understand the ACE binding and catalytic mechanism of action [1] Braz. J. Med. Biol. Res. (1981), 14, 173; [2] J. Am. Chem. Soc. (1993) 115, 11042;. Supported by Fapesp, CNPq and Capes.