Orientation of pyrenehexadecanoic acid (PyHA) on the water surface has been investigated using surface compression, fluorescence spectra, and two-photon ionization measurements. PyHA stays as a monomer at surface densities lower than 0.025 nmol/cm(2), while at high surface densities excimer emission was observed. The two-photon ionization signal had a peak at 90 degrees (p-polarized laser beam) upon the rotation of the polarization of the incident laser beam. The difference in the signal intensity generated by p-polarized and s-polarized lights became smaller as surface density increased. It is suggested that the tilt angle of the transition moment of the pyrene ring is ca. 70 degrees at a low surface density, and it increases with the surface density increase. Problems in determining orientation of the molecules on water are discussed.