CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is a zwitterionic surfactant, which has been extensively used in various biological fields. Ill the present work, the concentration-dependent aggregation Of the surfactant in deuterium Oxide solution was investigated by NMR spectroscopy. We have found that two break points exist oil the basis of the NMR parameters such as chemical shift difference (M), self-diffusion coefficient (D), and relaxation rates (R-1, R-2). The first break point corresponds to the widely accepted normal critical micelle concentration (cmc). The second one is Unexpected and ascribed to the second cmc, indicating that there is another type of micelle at higher concentrations. Further analysis Using I D selective NOESY with spin-diffusion quenching reveals that at the concentration above the second cmc CHAPS may form a two-layer spherical structure of micelles, with the aliphatic groups of CHAPS molecules in the inner layer interact with the steroid groups ill the outer layer. The existence of two types of micelles has also been supported by our TEM experiment. The dependence of CHAPS micelle size oil concentration explains why some proteins are soluble and stable only at concentrations above the second cmc. Therefore, our finding provides a basis For optimizing CHAPS concentration in functional and structural studies of membrane proteins.