Lipopolysaccharide (LPS) may cause sepsis when it enters the blood circulation. The toxic moiety of LPS is the well-preserved lipid A part. Lipid A contains two phosphate groups attached to diglucosamine, which are crucial for the many biological activities of LPS. In previous studies we found that alkaline phosphatase (AP) was able to dephosphorylate LPS. To test whether LPS-dephosphorylation can be used for intervention during sepsis, we investigated the effects of Salmonella minnesota Re 595 LPS and its dephosphorylated counterpart monophosphoryl lipid A (MPLA) on macrophage activation in vivo and in vitro. Exposure of RAW264.7 cells to LPS induced high levels of tumor necrosis factor (TNFalpha) and nitric oxide (NO), whereas MPLA elicited no response. LPS in vivo induced a significant rise in TNFalpha levels in mice and an enhanced inflammatory cell influx in the lung, whereas MPLA did not. Having shown the relevance of this particular phosphate group of LPS, we subsequently explored the LPS-dephosphorylating ability of AP in different tissues, and the effect of AP administration in mice challenged with LPS. Histochemical data show that AP dephosphorylated native LPS in all tissues examined, whereas MPLA was not dephosphorylated. When mice received AP immediately after the LPS challenge, the survival rate was 100%, over 57% in the control group. We conclude that the enzymatic removal of phosphate groups from LPS by AP represents a crucial detoxification reaction, which may provide a new strategy to treat LPS-induced diseases like sepsis.