Apolipoprotein AI (apoAI) is the principal protein constituent of high density lipoproteins and it plays a key role in human cholesterol homeostasis; however, the structure of apoAI is not clearly understood. To test the hypothesis that apoAI is organized into domains, three deletion mutants of human apoAI expressed in Escherichia coli were studied in solution and in reconstituted high density lipoprotein particles. Each mutant lacked one of three specific regions that together encompass almost the entire 243 aa sequence of native apoAI (apoAI ⌬44-126, apoAI ⌬139-170, and apoAI ⌬190-243). Circular dichroism spectroscopy showed that the ␣-helical content of lipid-free apoAI ⌬44-126 was 27% while the other mutants and native apoAI averaged 55 ؎ 2%, suggesting that the missing N-terminal portion contains most of the ␣-helical structure of lipid-free apoAI. ApoAI ⌬44-126 exhibited the largest increase in ␣-helix upon lipid binding (125% increase versus an average of 25% for the others), confirming the importance of the C-terminal half of apoAI in lipid binding. Denaturation studies showed that the N-terminal half of apoAI is primarily responsible for ␣-helix stability in the lipid-free state, whereas the C terminus is required for ␣-helix stability when lipid-bound. We conclude that the N-terminal half (aa 44-126) of apoAI is responsible for most of the ␣-helical structure and the marginal stability of lipid-free apoAI while the C terminus (aa 139-243) is less organized. The increase in ␣-helical content observed when native apoAI binds lipid results from the formation of ␣-helix primarily in the C-terminal half of the molecule.