The processing and release of 31-kDa proIL-1 beta to the mature 17-kDa form of IL-1 beta are still poorly understood. To help elucidate the mechanisms involved in IL-1 beta processing and release, we measured IL-1 beta forms released from endotoxin-stimulated monocytes by immunoprecipitation of [35S]methionine-labeled protein, by Western blots, and by our recently developed ELISA specific for proIL-1 beta. Our studies demonstrate that in addition to the 17-kDa mature IL-1 beta, IL-1 beta is also released as 31-, 28-, and 3-kDa molecules. The 31-kDa-released form of proIL-1 beta represented 20-40% of the total released IL-1 beta, as measured by SDS-PAGE with densitometry. This released proIL-1 beta was susceptible to ICE processing; however, this proIL-1 beta was not detectable by either a mature or proIL-1 beta-specific ELISA, suggesting that release induces a conformational change. The ELISA inability to detect proIL-1 beta was not due to inadequate sensitivity or subsequent degradation in the ELISA. Furthermore, while immunoaffinity-purified cytosolic proIL-1 beta could complex the type II IL-1R, released proIL-1 beta did not. Finally, the absence of a band shift in nondenaturing gel electrophoresis excluded proIL-1 beta binding to another protein. These findings imply that IL-1 beta is exported from monocytes as 3-, 17-, 28-, and 31-kDa forms and that the released 31-kDa form differs from cytosolic proIL-1 beta.