Interleukin-1beta (IL-1beta) is a master cytokine involved in initiating the innate immune response in vertebrates (Dinarello, C. A. (1994) FASEB J. 8, 1314-1325). It is first synthesized as an inactive 269-residue precursor (pro-interleukin-1beta or pro-IL-1beta). Pro-IL-1beta requires processing by caspase-1 to generate the active, mature 153-residue cytokine. In this study, we combined hydrogen/deuterium exchange mass spectrometry, circular dichroism spectroscopy, and enzymatic digestion comparative studies to investigate the configurational landscape of pro-IL-1beta and the role the N terminus plays in modulating the landscape. We find that the N terminus keeps pro-IL-1beta in a protease-labile state while maintaining a core region of stability in the C-terminal region, the eventual mature protein. In mature IL-1beta, this highly protected region maps back to the area protected earliest in the NMR studies characterizing an on-route kinetic refolding intermediate. This protected region also encompasses two important functional loops that participate in the IL-1beta/receptor binding interface required for biological activity. We propose that the purpose of the N-terminal precursor region in pro-IL-1beta is to suppress the function of the eventual mature region while keeping a structurally and also functionally important core region primed for the final folding into the native, active state of the mature protein. The presence of the self-inhibiting precursor region provides yet another layer of regulation in the life cycle of this important cytokine.