A series of enkephalin-like peptides (X-Tyr-Gly-Gly-R-Pro) were synthesized for assay of cathepsin L and papain. Enzymes acted only at the Gly-Gly bond to release N-terminal dipeptides. When X = dansyl and R = Phe(NO2) the substrate was suited for continuous fluorimetric assay of rat brain cathepsin L (Km 45 microM, kcat/Km 1333 mM-1 sec-1). The substituted pentapeptides provided information on the influence of P2, P2' residues on rates of Gly-Gly cleavage. The synthetic substrate provided rapid and sensitive assays for the brain cathepsin L and its interaction with 13-14 kDa (cerebrocystatin) and 70 kDa (T-kininogen) rat brain inhibitors. The suppression of cathepsin L- or papain-mediated hydrolysis of substrates by inhibitors may be the result of competition between their binding domains at the enzyme catalytic center.