Fluorescence microscopy, X-ray reflectivity and ellipsometry have been applied to the study of a monolayer of valine-gramicidin A (VGA) at the air-water interface to obtain insight into the conformation and orientation of VGA, and to clarify the shoulder observed in its pi-A curve (12.5-16 mN m(-1), 240-175 Angstrom(2) molecule(-1)). At low molecular areas, the X-ray reflectivities are consistent with formation of a single layer of tubular-shaped dimers. Fluorescence microscopy yielded homogeneous pictures suggesting that on the macroscopic scale the shoulder cannot be attributed to domain formation. Thicknesses calculated from the ellipsometric isotherm are consistent with the X-ray data.