The interaction of sphingosine (SP) and stearylamine (SA) with dipalmitoylphosphatidylserine (DPPS) has been studied by using differential scanning calorimetry (DSC) and phosphorus nuclear magnetic resonance (31P-NMR). DSC showed that SP and SA rigidified the membranes, forming an azeotropic mixture with DPPS. The azeotropic mixture which was formed between DPPS and SP was found at a DPPS/SP molar ratio of 2:1 whereas SA and DPPS formed an azeotropic mixture at a DPPS/SA molar ratio of 1:1. An eutectic point was observed at 85 mol% of SP and 90 mol% of SA in DPPS. 31P-NMR showed the presence of a lamellar phase at DPPS/SP and DPPS/SA molar ratios lower than 1:1, whereas at higher molar ratios and at high temperatures, besides the lamellar phase, an isotropic component was detected. It was found that, at physiological pH, both SP and SA were protonated in a large extent, i.e., positively charged, since their apparent pK in the membrane were 9.1 and 8.9, respectively. The results reported in this work may be relevant to understand a number of biological effects produced by these positively charged molecules, due to their electrostatic interaction with negatively charged phospholipids.