We have recently presented some sequence rules that govern the endoproteolytic processing of precursor proteins within the constitutive secretory pathway [Watanabe, T. et al. (1992) J. Biol. Chem. 267, 8270-8274; Watanabe, T. et al. (1993) FEBS Lett. 320, 215-218]. This cleavage seems to be catalyzed by furin, a mammalian homologue of the yeast processing endoprotease Kex2. In this study, not only by coexpression of furin and substrate precursors in a furin-deficient cell line but also by in vitro experiments using purified recombinant furin and substrate precursors, we determined the substrate specificity of furin. The sequence requirements of furin determined by the coexpression and in vitro experiments were essentially the same, and were consistent with the sequence rules for precursor cleavage within the constitutive secretory pathway.