The interaction of 1,2-dipalmitoylglycerol (1,2-DPG) with dipalmitoylphosphatidylserine (DPPS) has been studied in the presence and in the absence of Ca2+ by using differential scanning calorimetry (DSC) and 31P-nuclear magnetic resonance (31P-NMR). In the absence of Ca2+, DSC showed that 1,2-DPG increased the phase transition of DPPS, effect already noticed at very low 1,2-DPG concentrations, whereas lipid immiscibilities were detected at concentrations of 1,2-DPG higher than about 30 mol%. 31P-NMR indicated that lamellar phases were always present at concentration of 1,2-DPG lower than about 35 mol%, but at higher concentrations non-lamellar phases may be present in the fluid phase. As observed by DSC, the apparent pKa of the carboxyl group of DPPS was increased slightly in the presence of 1,2-DPG. In the presence of Ca2+, the effect of 1,2-DPG was to further increase the temperature of the onset of the phase transition, indicating an stabilization of the most rigid phase in the DPPS/1,2-DPG/Ca2+ samples. Even concentrations of 1,2-DPG as low as 1 mol% of the total lipid already produced a noticeable effect. Moreover, lipid immiscibilities were apparent at concentrations of 1,2-DPG higher than 20 mol%. Furthermore, the transition of the DPPS/Ca2+ complex observed by DSC at 155 degrees C was perturbed by the presence of 1,2-DPG, indicating a change in the structure of the crystalline complex. Interestingly, the effect of non-saturating Ca2+ concentrations on the DPPS phase transition was enhanced by the presence of 1,2-DPG. The effect reported here may be significant for a number of situations where Ca2+, phosphatidylserine and diacylglycerols are involved, such as fusion of membranes, where diacylglycerol may facilitate Ca(2+)-induced fusion, or the activation of enzymes such as protein kinase C and phospholipases.