Two cDNA clones corresponding to human microsomal dipeptidase (MDP, formerly referred to as dehydropeptidase-I or renal dipeptidase (EC 3.4.13.11] were isolated from human placental and renal cDNA libraries employing rapid amplification of cDNA ends strategy. The complete amino acid sequence deduced from the cDNAs contains 411 residues, beginning with a signal peptide of 16 residues. A highly hydrophobic sequence of 16 amino acids is located at the carboxyl terminus, supporting the previous observation which suggested that mature MDP is anchored to the membrane by covalently attached phosphatidylinositol. MDP has four potential N-glycosylation sites and has no apparent sequence similarity to other metallopeptidases. Expression of immunologically cross-reactive and enzymatically active MDP was attained in COS cells transfected with the cDNA. DNA and RNA blot analyses indicated the existence of a single gene and a substantial amount of 1.8-kilobase mRNA in kidney.