The mechanism of binding of two promising anticancer agents (the cytotoxic alkylphospholipids perifosine and miltefosine) to the Akt PH domain is investigated by high-resolution field-cycling (31)P nuclear magnetic resonance (NMR) spectroscopy using a spin-labeled recombinant PH domain. These results strongly indicate that there are two discrete amphiphile binding sites on the domain: (i) the cationic site that binds phosphoinositides and some alkylphospholipids and (ii) a second site that is occupied by only the alkylphospholipids. The identification of this second site for amphiphiles on the Akt1 PH domain provides a new target for drug development as well as insights into the regulation of the activity of the intact Akt1 protein. The field-cycling NMR methodology could be used to define discrete phospholipid or amphiphile binding sites on a wide variety of peripheral membrane proteins.