The family M1 of Zn‐dependent aminopeptidases comprises members of closely related enzymes which are known to be involved in a variety of physiologically important processes. On the basis of two highly conserved peptide motifs, we have identified a new member of this family by PCR amplification and cDNA‐library screening. The longest ORF encodes a protein of 930 residues. It contains the HEXXH(X)18E Zn‐binding motif and displays high homology to the other M1 family members except for its N‐terminus for which a signal sequence of 20 residues can be predicted. This interpretation was supported by expressing fusion proteins formed with green fluorescent protein which localized to intracellular vesicles in COS‐7 and BHK cells. Northern‐blot analysis revealed ubiquitous expression of a major 3.1‐kb transcript. For enzymatic studies, the complete protein was expressed in Sf 9 insect cells. When aminoacyl β‐naphthylamides were used as substrates, efficient hydrolysis was only observed for Leu (and to a lesser extent Met). The activity was inhibited by chelators of bivalent cations and by other known aminopeptidase inhibitors, but surprisingly puromycin was without effect. This newly identified puromycin‐insensitive leucyl‐specific aminopeptidase is a signal‐sequence‐bearing member of family M1 and may be another example of the small subset of substrate‐specific peptidases.