It has previously been shown that rat liver microsomes contain a proteolytic enzyme that cleaves honeybee prepromelittin to yield promelittin. This enzyme has now been further purified by centrifugation on a sucrose-deoxycholate gradient and then reconstituted into phospholipid vesicles. Incubation of prepromelittin with vesicles in the presence of melittin yields, in addition to promelittin, a hydrophobic peptide. The latter could be isolated by extraction with l-butanol and paper electrophoresis in 30% formic acid and was shown to be intact signal peptide by analysis of peptic fragments and automated Edman degradation. The microsomal enzyme is thus an endoprotease that hydrolyzes prepromelittin exclusively at the pre-pro junction. The precision of this cleavage of an insect preprotein by a rat liver enzyme indicates that we are dealing with the ubiquitous eukaryotic signal peptidase.