Proprotein Convertases (PC) form an important class of serine endopeptidases implicated in many physiological and pathological processes. Furin (E.C. 3.4.21.75), is 96 kDa Ca²⁺ dependent endopeptidase, and is one of these PCs that activates a large numbers of proprotein through controlled cleavage in the cell secretory pathway compartments after the general motif (K/R)–Xn–(K/R)–R¯, where X indicates any amino acid residue less cysteine, and n is the number of spacer amino acid residues (1 or 3). We have synthesized series of FRET peptides containing these motifs obtained from the sequence of proproteins, prohormones, and proteins of pathogenic agents to examine the hydrolysis of them by hFurin. The kinetic data shows that some sequences without Arg residue in P4 were poorly hydrolyzed, showing an increase in Km values at 9 times. Although in P3 the Glu or Gln residues increase the Km values, the Arg residues in same position have an opposite effect. Leu, Val and Ile residues do not appear in any peptide sequence in P1' subsite. We also observed that peptides containing Ser or Asp in P1' and Val in P2' positions were the most efficiently cleaved substrates. Among the 100 assayed FRET peptides, Abz-HHRQRRSVSIQ-EDDnp was observed to be the best substrate so far described for hFurin, being hydrolyzed with the kinetic parameters Km= 1.51 mM, kcat= 89 s^-1, and kcat/Km = 58808 (mM.s)^-1. The study of specific requirements of each enzyme subsite in natural substrates is in progress and will be also presented.