Cathepsin S is a lysosomal cysteine peptidase that is thought to play an important role in the processing of histocompatibility complex (MHC) class II-associated invariant chain. The functional importance of the enzyme has been extensively explored however, the exact role of cathepsin S needs to be defined. Cathepsin S is the unique among lysosomal cathepsins by being highly active and stable also at neutral pH. This characteristic suggests that the enzyme may be involved in extracellular processes, being the ionic strength and the stability of the enzyme important for the enzyme activity out of the lysosomes. The aim of our work was to study the S₂ to S₁' subsites specificity of cathepsin S using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=N-[2,4-dinitrophenyl] ethylenediamine). We assayed three series of peptides, namely Abz-KLXSSKQ-EDDnp, Abz-KXRSSKQ-EDDnp and Abz-KLRXSKQ-EDDnp (X= various amino acids). The results indicated that S₁ presents a broad specificity accepting positively charged, (Arg, Lys), polar non-charged (Met) and hydrophobic aliphatic (Leu, Val) residues. The subsite S₂ is more restrict preferring hydrophobic aliphatic residues, (Leu and Val). The subsite S₁' accommodates well hydrophobic aliphatic residues being the peptide Abz-KLRLSKQ-EDDnp hydrolyzed with the highest catalytic efficiency in all the series. A significant activation of Abz-KLRSSKQ-EDDnp hydrolysis by cathepsin S was promoted in presence of 2.5 M NaCl, being the activity almost 9-fold higher than in the absence of the salt. In addition, we developed FRET peptides derived from putative natural cathepsin S substrates as invariant chain, insulin β-chain and amyloid β peptides. The peptide Abz-ATPLLMQ-EDDnp, derived from invariant chain, assayed in 50mM sodium phosphate and 2.5mM EDTA, pH 7.5, in presence of 1M NaCl, was well hydrolyzed by cathepsin S and almost resistant to cathepsins L, V, K and B. These conditions can be important to differentiate cathepsin S among other cathepsins and for the development of specific substrates for the enzyme. (Supported by FAPESP and CNPq)