It is postulated that biological membrane lipids are heterogeneously distributed into lipid microdomains. Recent evidence indicates that docosahexaenoic acid-containing phospholipids may be involved in biologically important lipid phase separations. Here we investigate the elastic and thermal properties of a model plasma membrane composed of egg sphingomyelin (SM), cholesterol and 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE). Two techniques are employed, pressure-area isotherms on monolayers to examine condensation and interfacial elasticity behavior, and differential scanning calorimetry (DSC) on bilayers to evaluate phase separations. Significant levels of condensation are observed for mixtures of SM and cholesterol. Surface elasticity measurements indicate that cholesterol decreases and SDPE increases the in-plane elasticity of SM monolayers. At X(SDPE)> or =0.15 in SM, a more horizontal region emerges in the pressure-area isotherms indicating 'squeeze out' of SDPE from the monolayers. Addition of cholesterol to equimolar amounts of SM and SDPE further increases the amount of 'squeeze out', supporting the concept of phase separation into a cholesterol- and SM-rich liquid ordered phase and a SDPE-rich liquid disordered phase. This conclusion is corroborated by DSC studies where as little as X(Chol)=0.0025 induces a phase separation between the two lipids.