Little is known on the forms of interleukin-1β (IL-1β) that are produced by microglial cells in the nervous system. Mixed glial cell cultures of rats produced IL-1β in response to lipopolysaccharide (LPS). Using Western blot, pro-IL-1β was found to be localized both intracellularly and in the supernatant, whereas mature IL-1β was found only in the supernatant but in lower quantities than pro-IL-1β. Immunocytochemistry confirmed that microglial cells are the exclusive source of IL-1β. Blockade of the IL-1β-converting enzyme (ICE) by Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO) decreased the levels of mature IL-1β but had no effect on pro-IL-1β. Release of pro-IL-1β was not associated with cell death nor with the extracellular release of ICE. Using gelatin zymography, glial cells were found to express constitutive matrix metalloproteinases (MMP) in the form of MMP-2. Exposure to LPS induced MMP-9 expression in a time-dependent manner similar to the pro-IL-1β expression profile. MMP activation and inhibition experiments indicated a possible role of MMPs in the cleavage of pro-IL-1β but not in the generation of mature IL-1β. Microglial cells share with macrophages the ability to release large amounts of pro-IL-1β of which the extracellular role remains to be determined.