It is presently unclear what role elevations in intracellular calcium concentration ([Ca2+]i) play in the control of monokine secretion, or whether such alterations underlie the ability of physiologic stimuli to induce production of these important signalling molecules. To address these issues, we have performed experiments in murine peritoneal macrophages to determine whether lipopolysaccharide (LPS) or interferon gamma (IFN-gamma) initiate production of the proinflammatory monokine interleukin 6 (IL-6) concomitant with elevations in [Ca2+]i and with kinetics similar to that seen with known Ca2+ mobilizing agents. Alterations in [Ca2+]i after treatment with LPS, IFN-gamma, platelet activating factor (PAF), or thapsigargin were measured by fluorimetric methods. These effects were compared with the ability of each to induce IL-6 mRNA expression as measured by semiquantitative reverse-transcribed polymerase chain reactions. We report that neither LPS nor IFN-gamma elicited detectable elevations in [Ca2+]i but that both up-regulated expression of IL-6 mRNA expression within 60 min. In contrast, experiments using either thapsigargin or PAF showed rapid and dramatic elevations in [Ca2+]i with marked increases in IL-6 mRNA expression, as quickly as 15 min after initial exposure. Elevations in mRNA encoding IL-6 by thapsigargin and PAF were found to occur in a dose-dependent manner, mirroring their ability to elicit elevations in [Ca2+]i. These data demonstrate that LPS and IFN-gamma induce IL-6 message expression by means of Ca2+-independent signalling pathways. Furthermore, Ca2+-mobilizing agents that evoke monokine message expression do so far more rapidly than do LPS or IFN-gamma. Taken in concert, these data are consistent with the hypothesis that multiple signalling pathways exist by which production of proinflammatory monokines are initiated.