The proprotein convertases are believed to be responsible for the proteolytic maturation of a large number of peptide hormone precursors. Although potent furin inhibitors have been identified, thus far, no small-molecule prohormone convertase 1/3 or prohormone convertase 2 (PC2) inhibitors have been described. After screening 38 small-molecule positional scanning libraries against recombinant mouse PC2, two promising chemical scaffolds were identified: bicyclic guanidines, and pyrrolidine bis-piperazines. A set of individual compounds was designed from each library and tested against PC2. Pyrrolidine bis-piperazines were irreversible, time-dependent inhibitors of PC2, exhibiting noncompetitive inhibition kinetics; the most potent inhibitor exhibited a K(i) value for PC2 of 0.54 microM. In contrast, the most potent bicyclic guanidine inhibitor exhibited a K(i) value of 3.3 microM. Cross-reactivity with other convertases was limited: pyrrolidine bis-piperazines exhibited K(i) values greater than 25 microM for PC1/3 or furin, whereas the K(i) values of bicyclic guanidines for these other convertases were more than 15 microM. We conclude that pyrrolidine bis-piperazines and bicyclic guanidines represent promising initial leads for the optimization of therapeutically active PC2 inhibitors. PC2-specific inhibitors may be useful in the pharmacological blockade of PC2-dependent cleavage events, such as glucagon production in the pancreas and ectopic peptide production in small-cell carcinoma, and to study PC2-dependent proteolytic events, such as opioid peptide production.