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Several specimens of anhydrous ampicillin were prepared by heating the ampicillin trihydrate at 100, 120, 140 and 160 °C. The effects of dehydration temperature on water vapor adsorption, dissolution behavior and surface property were investigated. The water vapor adsorption of anhydrous ampicillin was studied at 89% relative humidity, 40 °C and the water vapor adsorption rate was found to decrease with increase of dehydration temperature. Dissolution profiles of the various anhydrous specimens were investigated in 96% ethanol at 35 °C by the static disk method. The anhydrous form prepared at higher dehydration temperature exhibited faster dissolution rate. Solid phase transformation from the anhydrous form to the trihydrate form occurred during the dissolution test. The rate of phase transformation during the dissolution test decreased with increasing dehydration temperature. Topographic difference of the anhydrous forms prepared at 100 and 160 °C was not observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM); however, difference of the microstructural properties was apparently observed by the AFM phase image. Surface free energy study revealed that when ampicillin was dehydrated at high temperature, the sample surface became more hydrophobic resulting in less interaction force with water and slow water sorption rate. From the results, we concluded that the polarity of sample surface induced by dehydration of ampicillin would affect the phase transformation and dissolution behavior.