Hydrothermal modification of the alumina catalyst of n-butenes skeletal isomerization was investigated. It is shown that during the hydrothermal treatment of gamma-Al2O3 and subsequent calcination its activity in skeletal isomerization of n-butenes first increases, and then it decreases with a rise of the hydrothermal treatment duration. This behavior is due to a similar change in the content of the strong Lewis acid sites of alumina, which are the active centers of reaction and they can be identified by IR-spectroscopy of the adsorbed pyridine and EPR-spectroscopy of the adsorbed anthraquinone. We report the mechanism of new Lewis acid sites formation at the gamma-Al2O3 hydrothermal treatment containing X-ray amorphous component.