Purified calpain II from vascular smooth muscle is a heterodimer consisting of catalytic (Mr = 76,000) and regulatory (Mr = 30,000) subunits. In the presence of Ca2+, the regulatory subunit undergoes stepwise autolysis resulting in enzyme activation. By slowing autoproteolysis, we identified major autolytic intermediates of the regulatory subunit. Gas-phase sequencing of the regulatory subunit and its autolytic fragments revealed that the NH2-terminus of the Mr = 30,000 form was blocked, whereas each fragment yielded a unique amino acid sequence, suggesting that autolysis proceeds in an NH2- to COOH-terminal direction. By comparison of actual amino acid sequences of autolytic cleavage intermediates to the full sequence deduced from cDNA, we have identified the major autolytic cleavage sites. Three different peptide bonds were cleaved, with neutral amino acids predominating on both sides of the peptide bond hydrolyzed. Importantly, leucine or isoleucine was identified in the second position upstream from the cleavage site in all three autolytic sequences. The presence of an upstream leucine residue in the autolytic cleavage sequence is reminiscent of the structure of potent microbial and synthetic peptide inhibitors of calpain.