Previous studies have shown that the prohormone convertase 1 (PC1, or SPC3), a member of the new eukaryotic subtilisin-like proteinase family, undergoes a series of proteolytic processing events during its biosynthesis. The first cleavage, of the amino-terminal prosegment, is probably involved in enzyme activation, while the secondary cleavages at the carboxyl terminus are of unknown significance and occur mainly in cells possessing a regulated secretory pathway. In this work, we found that 87-kDa PC1, a homogeneous recombinant protein, could spontaneously convert to 74- and 66-kDa forms in vitro. Limited digestion of 87-kDa PC1 using chymotrypsin and trypsin could also generate 74- and 66-kDa-like PC1s, which were enzymatically active against the fluorogenic peptide carbobenzoxy-Arg-Tyr-Lys-Arg-aminomethylcoumarin. The 74/66-kDa PC1 generated by spontaneous conversion was purified away from the 87-kDa form and enzymatically characterized. Compared to the 87-kDa form, 74/66-kDa PC1 was more active but less stable. In addition, 74/66-kDa PC1 exhibited a narrower pH optimum (between 5.0 and 5.5) and was activated by higher concentrations of calcium. Carboxyl-terminally truncated PC1 also appeared to be more sensitive to certain protease inhibitors than 87-kDa PC1. Taken together, our results suggest that autocatalysis could be involved in carboxyl-terminal cleavages of PC1. These carboxyl-terminal cleavages of PC1 result in alterations in certain PC1 properties and may therefore possess potential significance with respect to prohormone processing.