The phase diagram of the DSPC/DOPC/cholesterol biomembrane model system was recently determined by studies in our laboratory. However, the component DOPC does not occur in animal cell plasma membranes, but was chosen for convenience in solving the phase diagram. In my study, DOPC was replaced stepwise by the naturally-occurring SOPC to determine its effect on the high cholesterol boundary of the L?+Lo two-phase region. Giant unilamellar vesicles with the chosen compositions, probed with the fluorescent dye C12:0-DiI, were made by electroswelling and their phase behaviors were examined by fluorescence microscopy. From 0 to 45% replacement of DOPC by SOPC, the middle part of the boundary shifts only a small amount, from cholesterol mole fraction 0.38 down to mole fraction 0.30 ? 0.35. The L? side of the boundary remains essentially unchanged. Above 50% replacement, photo-induced artifacts obscure the real phase behavior at the middle part of the boundary, but might represent the actual boundary termination. Above 70% replacement of DOPC by SOPC, no phase-separation was observed for the L? side of the boundary. The size and shape of the L?+Lo two-phase region can be related to the structures of the lipids.