The effect of pressure on bovine alpha-lactalbumin (LA) has been investigated by fluorescence methods. The intrinsic fluorescence spectra of holo-LA (CaII-bound LA) hardly changed in its intensity and maximum wavelength on increasing the pressure up to 400 MPa. In the intrinsic fluorescence spectrum of apo-LA (CaII-depleted form) the maximum wavelength was red-shifted, and the intensity was increased to a large extent by increasing pressure. The fluorescence titrations of both forms of LA were performed with a fluorescent hydrophobic probe 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonate (bis-ANS) at various pressures, and binding constants (Kb) of bis-ANS were calculated. The Kb-value for holo-LA slightly decreased from 0.1 to 100 MPa and increased above 200 MPa. The Kb value for apo-LA gradually increased with increasing pressure up to 400 MPa. These results were explained by the difference in hydrophobic characteristics of holo- and apo-LA.