Diacetylpolyamines (DAPs) are novel, promising tumor related markers, but the mechanism sustaining their good sensitivity and specificity is not known. This investigation was conducted on the production mechanism of DAPs, using (C57BL/6N×DBA/2N)F₁ mice and the P388D₁ (lymphoid) tumor system. Spleen adherent cells from mice injected with thioglycollate produced N¹,N¹²-diacetylspermine (DAM) in co-culture with P388D₁ cells. Macrophages among peritoneal exuded cells also produced DAM actively, while granulocytes, another innate immune cell, did not. The participation of macrophages in vivo was confirmed by depletion experiments using dichloromethylene diphosphonate liposomes. The supply of exogenous spermine and a deficiency of glucose, which tend to occur with tumorigenesis, resulted in an explosion of the production of DAM by macrophages. The number of macrophages required for the elevation of DAM for a diagnosis in humans was estimated at less than 4.2×10⁸. The mechanism and productivity studied in this work support the superiority of DAM as a tumor related marker. Diacetylation may relate to the depression of macrophage function by spermine.