The proteolytic generation of mature interleukin-1 beta (IL-1 beta) from its inactive precursor does not proceed by a conventional pathway for hormonal processing. Pro-IL-1 beta is found dispersed in the cytoplasm, and there are no basic amino acid residues or other commonly recognized processing sites adjoining the mature N-terminus. Processing appears to occur during release of the hormone. In the present study, we have identified a specific protease that generates mature IL-1 beta from the precursor. This enzyme is co-induced with the hormone, and it differs in its cleavage specificity and inhibitor sensitivity from all known proteases.