Treatment of resident peritoneal macrophages of rats with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV) potentiated their activation for tumor cell lysis by endotoxins. The fluorescence polarization of diphenylhexatriene (DPH) embedded in rough endoplasmic reticulum membranes isolated from DPPC SUV-treated macrophages was enhanced. The average fluorescence lifetime of DPH and the rotational correlation time deduced from anisotropy decay were unchanged, whereas the residual anisotropy and hence the order parameter were increased. The measurement of the fluorescence anisotropy of DPH as a function of the temperature showed a phase transition. No phase transition was observed in the rough endoplasmic reticulum membranes of macrophages either treated or not treated with cholesterol/DPPC SUV (1/1; mol/mol). The synergistic effect of DPPC SUV on the tumoricidal activity of macrophages induced by endotoxins appears to be correlated with the changes in the properties of the rough endoplasmic reticulum membranes. Both effects were transient; they had the same kinetics of induction and reversion, and they were both inhibited by cholesterol.