We study the effect of Ca2+ on the lateral segregation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). Supported lipid bilayers (SLBs) were observed by atomic force microscopy (AFM). Since SLBs are formed from liposomes of POPE:POPG, we examined the effect of calcium on these suspensions by differential scanning calorimetry (DSC) and 31P nuclear magnetic resonance spectroscopy (31P NMR). AFM images revealed the existence of two separated phases, the higher showing a region with protruding subdomains. Force spectroscopy (FS) was applied to clarify the nature of each phase. The values of breakthrough force (F y), adhesion force (Fadh), and height extracted from the force curves were assigned to the corresponding gel (Lβ) and fluid (Lα) phase. The endotherms obtained by DSC suggest that, in the presence of Ca2+, phase separation already exists in the suspensions of POPE:POPG used to form SLBs. Due to the temperature changes applied during preparation of SLBs a 31P NMR study was performed to assess the lamellar nature of the samples before spreading them onto mica. With in situ AFM experiments we showed that the binding of Ca2+ to POPG-enriched domains only induces the formation of subdomains in the L β phase. © 2009 American Chemical Society.