Alkylglycerols, inflammation products of cancerous tissues, are potent macrophage activating agents. A brief in vitro treatment (30 min) of mouse peritoneal cells with a low concentration (50 ng/mL) of dodecylglycerol (DDG) in 10% foetal calf serum supplemented RPMI-1640 medium (FCS medium) activates macrophages for Fc-receptor mediated ingestion activity. A serum factor(s) was shown to be required for the activation of macrophages. When non-adherent cells were treated with rac-sn-1(3)-dodecylglycerol (DDG) in a serum free-0.1% egg albumin supplemented RPMI medium (EA medium) for 30 min and cultured in FCS medium for 2 h, the resultant conditioned medium contained a signal factor able to activate macrophages (macrophage activating factor). A conditioned medium prepared with electrophoresed serum alpha 2-globulin fraction in EA medium markedly enhanced activation of macrophages. Incubation of DDG-treated non-adherent cell ghosts in EA medium containing alpha 2-globulin also produced the macrophage activating signal factor. Therefore, it is concluded that a serum factor in alpha 2-globulin fraction is processed by pre-existing functions or enzymes of DDG-treated non-adherent cell membrane to yield a macrophage activating signal factor.