The macrophage-suppressive properties of three bisphosphonates were evaluated by studying their effect on nitric oxide (NO) production by activated RAW 264 macrophage-like cells. The cells were activated with 10 micrograms/ml of lipopolysaccharide, and NO was determined as nitrite in the cell culture supernatant. The effect of the drugs on inducible NO synthase was determined by Western blot analysis. As free drugs, clodronate and pamidronate inhibited NO secretion in a dose-dependent manner, whereas alendronate had no effect. Liposome encapsulation enhanced the effect of clodronate by a factor of 7, but the potency of pamidronate weakened slightly when encapsulated in liposomes. The inducible NO synthase expression inside the cells was also decreased by liposomal clodronate. In contrast to pamidronate, clodronate could affect the NO secretion when given to the cells simultaneously with lipopolysaccharide, and the inhibitory action was still seen when the drug was added 2 h after lipopolysaccharide induction. The viability of the cells was not affected by free or liposomal clodronate, whereas pamidronate showed considerable cytotoxicity. This study shows the different actions of these three bisphosphonates on NO production by macrophages and suggests that liposomal clodronate is the most promising bisphosphonate as an anti-inflammatory agent, whereas aminobisphosphonates do not possess anti-inflammatory properties.