LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A2 (PLA2) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i ) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15-20 Å relative to PLA2. ii ) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii ) Aβ -hairpin resembling a lipase lid separates S181 from solvent. iv ) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specifi city, and K128 and R147, whose mutations cause LCAT defi ciency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport. -Segrest, J. P., M. K. Jones, A. Catte, and S. P. Thirumuruganandham. A robust all-atom model for LCAT generated by homology modeling.