The specificity of the S1' subsite of the cysteine proteases cathepsin B, L, S and papain has been investigated using a series of intramolecularly quenched fluorogenic substrates (Dansyl-Phe-Arg-AA-Trp-Ala) where the P1' amino acid (AA) has been varied. Taken individually, each enzyme displays a relatively broad S1' subsite specificity and this subsite cannot be considered as a primary site of specificity. Notable differences do exist however between the various proteases. Cathepsin B prefers large hydrophobic residues in the P1' position of a substrate while cathepsin L has an opposite trend, favoring amino acids with small (Ala, Ser) or long but non-branched (Asn, Gln, Lys) side chains. Cathepsin S and papain display a somewhat broader S1' subsite specificity.