The primary structures of porcine and human pancreatic elastase II precursors were elucidated by molecular cloning and cDNA sequence analysis. The sequences of the cDNAs cloned from a human pancreatic cDNA library indicate that at least two elastases II are expressed in this tissue. These two human elastases II have been designated elastases IIA and IIB. All the cDNA sequences obtained, including porcine elastase II cDNA, reveal that elastase II is synthesized as a preproenzyme of 269 amino acids, including a predicted signal peptide of 16 amino acids and a predicted activation peptide of 12 amino acids. Human elastase IIA contains the published amino-terminal sequence (16 residues) for human pancreatic proelastase II, whereas elastase IIB shares 50% homology with the published 16-residue sequence. However, there is 90% homology between the overall amino acid sequences of elastases IIA and IIB. Blot hybridization analysis of the polyadenylated RNAs isolated from various human tissues demonstrates that the human elastase II mRNAs are specifically detected in the pancreas.