Nitrogen-containing bisphosphonates (NBPs), anti-bone-resorptive drugs, exhibit inflammatory side effects (fever, jaw osteomyelitis or osteonecrosis, etc.). We previously reported that in mice: (i) a single intraperitoneal injection of alendronate (an NBP, 40 μmol/kg or less) induces various inflammatory reactions, (ii) these effects, which are minimal in IL-1-deficient mice, can be prevented by co-administration of clodronate (a non-NBP, 40 μmol/kg or less), and (iii) alendronate increases IL-1β in tissues (liver, spleen, and lung), but strangely not in blood. Here, we found the following in mice. (a) The IL-1β in tissues is pro-IL-1β. (b) Unlike LPS, alendronate induces minimal activation of caspase-1 (pro-IL-1β-processing enzyme). (c) The tissue pro-IL-1β elevations are largely absent in macrophage-depleted mice. (d) In vitro, 100 μM alendronate directly stimulates RAW 264 cells (murine macrophage-like cells) to produce pro-IL-1β, and 1 μM clodronate inhibits this effect. These results suggest that in mice: (i) the major pro-IL-1β-producing cells in response to alendronate are macrophages, (ii) alendronate directly stimulates them to produce pro-IL-1β, but the release of mature IL-1β is below detectable levels due to insufficient activation of caspase-1, and (iii) clodronate inhibits the pro-IL-1β production by acting directly on macrophages, although the in vivo mechanism may differ from the in vitro one.