In model membranes phospholipids (PLs) with high and low transition melting temperatures (Tm) may segregate laterally, especially in the presence of cholesterol. Cholesterol prefers interacting with PLs with saturated acyl chains over those with monounsaturated and especially polyunsaturated acyl chains. In addition to unsaturation, the headgroup and the phospholipid backbone (glycerol versus sphingosine) will also affect how cholesterol interacts with the PLs. Likely these interactions between cholesterol and different phospholipids plays a role in the formation of lateral membrane structure. In this project we study how cholesterol promotes the segregation of low and high Tm lipids into ordered and disordered domains.We have determined the formation of liquid-ordered phase with time-resolved fluorescence spectroscopy of trans-parinaric acid (tPA) in ternary bilayers containing cholesterol, palmitoyl-sphingomyelin (PSM) and different unsaturated PLs. The results were compared to results from cholesterol-free bilayers.The results showed that cholesterol lowered the amount of PSM needed to form liquid-ordered phase (Lo) compared to gel phase formation in cholesterol free bilayers. When the number of double bonds increased in the low Tm lipid, even lower amounts of PSM was needed to form Lo. We will further investigate if cholesterol have the same effect on formation of ordered phases when the high Tm lipid is varied and determine how the domain properties changes as a consequence.In conclusion, cholesterol enhances phase separation of low and high Tm lipids. The aversion of cholesterol for polyunsaturated PLs further enchases the phase separation. Altogether, this supports an important role of cholesterol to maintain and regulate lateral structure in biological membranes.