The mechanisms underlying caspase-1 activation and IL-1beta processing during inflammatory activation of monocytes and macrophages are not well defined. Here, we describe an in vitro proteolytic processing assay that allows for comparison of caspase-1 regulatory components in a cell-free system separately from the confounding issue of IL-1beta secretion. Analysis of in vitro IL-1beta and caspase-1 processing in lysates from unstimulated Bac1 murine macrophages indicated a slow rate of basal caspase-1 activation and proteolytic maturation of IL-1beta. In contrast, brief (5 min) treatment of intact macrophages with extracellular ATP (as an activator of the P2X(7) receptor) or nigericin before cell lysis markedly accelerated the in vitro processing of caspase-1 and IL-1beta. This acceleration of in vitro processing was strictly dependent on loss of intracellular K(+) from the intact cells. The induction of in vitro caspase-1 activation by lysis per se or by K(+) loss before lysis was sensitive to pretreatment of intact macrophages with the tyrphostin AG-126 or bromoenol lactone, an inhibitor of Ca(2+)-independent phospholipase A(2). Caspase-1 activation and IL-1beta processing in lysates from unstimulated macrophages were also accelerated by addition of recombinant ASC, a previously identified adapter protein that directly associates with caspase-1. These data indicate that increased K(+) efflux via P2X(7) nucleotide receptor stimulation activates AG-126- and bromoenol lactone-sensitive signaling pathways in murine macrophages that result in stably maintained signals for caspase-1 regulation in cell-free assays.