The serotonin transporter (SERT) is the target of several important antidepressant and psychostimulant drugs. It has been shown that under defined conditions, the transporter spread at the air/water interface was able to bind its specific ligands. In this paper, the interfacial organization of the protein has been assessed from dynamic surface pressure and ellipsometric measurements. For areas comprising between 10,400 and 7,100 A(2)/molecule, ellipsometric measurements reveal an important change in the thickness of the SERT film. This change was attributed to the reorientation of the transporter molecules from a horizontal to their natural predictive transmembrane orientation. The thickness of the SERT film at 7,100 A(2)/molecule was found to be approximately equal to 84 A and coincided well with the theoretical value estimated from the calculations based on the dimensions of alpha-helices containing membrane proteins. These data suggest that the three-dimensional arrangement of the SERT may be represented as a box with lengths d(z)=83--85 A and d(y) or d(x)=41--47 A.