We describe quantitatively the interactions in a mixture of a saturated and an unsaturated phospholipid, and their consequences to the phase behavior at macroscopic and microscopic levels. This type of lipid-lipid interaction is fundamental in determining the organization and physical behavior of biological membranes. Mixtures of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) are examined in detail by multiple experimental approaches (differential scanning calorimetry (DSC), fluorescence resonance energy transfer, and confocal fluorescence microscopy) in combination with Monte Carlo simulations in a lattice. The interactions between all possible pairs of lipid species and states are determined by matching the heat capacity calculated through Monte Carlo simulations to that measured experimentally by DSC. Only for one other lipid system, a mixture between two saturated phosphatidylcholines, is a similar quantitative description available. The interactions in the two systems and different representations used to model them are compared. Phase separation occurs in DPPC/POPC at about the center of the phase diagram mapped by DSC, but not at all compositions and temperatures in the coexistence region. Close to the extremes of composition, the phase behavior is best described by large fluctuations. At the heat capacity maxima in the mixtures, the domain size distributions change remarkably; large domains disappear and cooperative fluctuations increase.