In this work, the surface and fluorescent behavior of three phospholipids containing a pyrene molecule in one of their hydrocarbon chains was studied. Differences between the isotherms provided by the different monolayers can be attributed to the orientation of the head group in the phospholipids at the air/water interface. This assertion is supported by the fluorescent behavior of monolayers of phospholipids containing labeled pyrene (Py-DPPE, Py-DPPC, and Py-DPPG).