Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. PC2 and furin are two recently identified members of a class of mammalian proteins homologous to the yeast precursor processing protease kex2 and the bacterial subtillisins. We have used the polymerase chain reaction to identify and clone a cDNA (PC3) from the mouse AtT20 anterior pituitary cell line that represents an additional member of this growing family of mammalian proteases. PC3 encodes a 753-residue protein that begins with a signal peptide and contains a 292-residue domain closely related to the catalytic modules of PC2, furin, and kex2. Within this region 58%, 65%, and 50% of the amino acids of PC3 are identical to those of the aligned PC2, furin, and kex2 sequences, respectively, and the catalytically important Asp, His, and Ser residues are all conserved. On Northern blots, PC3 hybridizes to two transcripts of 3 and 5 kilobases. Tissue distribution studies indicate that both PC2 and PC3 are expressed in a variety of neuroendocrine tissues, including pancreatic islets and brain, but are not expressed in liver, kidney, skeletal muscle, and spleen. The high degree of similarity of PC3, PC2, and furin suggests that they are all members of a superfamily of mammalian proteases that are involved in the processing of prohormones and/or other protein precursors. In contrast to furin, PC3, like PC2, lacks a hydrophobic transmembrane anchor, but it has a potential C-terminal amphipathic helical segment similar to the putative membrane anchor of carboxypeptidase H. These and other differences suggest that these proteins carry out compartmentalized proteolysis within cells, such as processing within regulated versus constitutive secretory pathways. 