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We performed a series of mutations in the human apolipoprotein A-I (apoA-I) gene designed to alter specific amino acid residues and domains implicated in lecithin: cholesterol acyltransferase (LCAT) activation or lipid binding. We used the mutant apoA-I forms to establish nine stable cell lines, and developed strategies for the large scale production and purification of the mutated apoA-I proteins from conditioned media.HDL 162 3 Leu,Leu 189 3 Trp) exhibited normal LCAT activation as compared with the wild type proapoA-I and plasma apoA-I forms. The apparent catalytic efficiency (V max(app) /K m(app) ) of the apoA-I mutants ranged from 17.8 to 107.2% of the control and was the result of variations in both the K m and the V max in the different mutants. These findings indicate that putative helices 6 and 7, and the carboxyl-terminal helices 8 and 9 contribute to the optimum activation of lecithin:cholesterol acyltransferase. In addition to their use in the present study, the variant apoA-I forms generated will serve as valuable reagents for the identification of the domains and residues of apoA-I involved in binding the scavenger receptor BI, and facilitating cholesterol efflux from cells as well as aid in the structural analysis of apoA-I.