IL-1β released from activated macrophages contributes significantly to tissue damage in inflammatory, degenerative, and autoimmune diseases. In the present study, we identified a novel mechanism of IL-1β release from activated microglia (brain macrophages) that occurred independently of P2X 7 ATP receptor activation. Stimulation of LPS-preactivated microglia with lysophosphatidylcholine (LPC) caused rapid processing and secretion of mature 17-kDa IL-1β. Neither LPC-induced IL-1β release nor LPC-stimulated intracellular Ca 2+ increases were affected by inhibition of P2X 7 ATP receptors with oxidized ATP. Microglial LPC-induced IL-1β release was suppressed in Ca 2+-free medium or during inhibition of nonselective cation channels with Gd 3+ or La 3+. It was also attenuated when Ca 2+-activated K+ channels were blocked with charybdotoxin (CTX). The electroneutral K+ ionophore nigericin did not reverse the suppressive effects of CTX on LPC-stimulated IL-1β release, demonstrating the importance of membrane hyperpolarization. Furthermore, LPC-stimulated caspase activity was unaffected by Ca 2+-free medium or CTX, suggesting that secretion but not processing of IL-1β is Ca 2+-and voltage-dependent. In summary, these data indicate that the activity of nonselective cation channels and Ca 2+-activated K+ channels is required for optimal IL-1β release from LPC-stimulated microglia.