The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type ( cis vs. trans ), or position of the double bonds in 18 or 20 carbon sn -2 fatty acyl chains and recombined with [ 3 H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn -2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn -2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon ( r 2 ؍ 0.61, n ؍ 18) and 20 carbon ( r 2 ؍ 0.93, n ؍ 5) sn -2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn -2 fatty acyl chain PC species and 90% of an inert PC ether matrix ( sn -1 18:1, sn -2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated ( r 2 ؍ 0.71) with that of 100% PC rHDL containing the same 18 carbon sn -2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity.We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant. -Parks,