A search for low molecular weight peptide substrates for the metalloendoproteinase, human fibroblast stromelysin, resulted in the discovery that substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) is a substrate for this enzyme and is cleaved exclusively at the Gln6-Phe7 bond. On the basis of this observation, a semicontinuous HPLC-based assay was developed that monitors the production of the hydrolysis product, fragment 7-11 (SP7-11). Steady-state velocities for the production of SP7-11 have been determined as a function of substrate concentration and obey simple, Michaelis-Menten kinetics. For a 1-ml reaction volume, Vmax = (2.4 nmol SP7-11/min)/micrograms protein and Km = 0.38 mM.