Pulsed electric fields in combination with vacuum infusion have been utilized to impregnate cells with trehalose, aiming at substantially improving the freezing tolerance of spinach leaves. Spinach samples were first treated with ten trains of bi-polar, rectangular electric field pulses with a nominal electric field strength of 580 V/cm and immediately immersed in a 40% (w/w) solution of trehalose under vacuum for 20 min. The samples were kept in the trehalose solution for 2.5 h at atmospheric pressure, immersed in deionised water at 4 degrees C overnight, frozen in liquid nitrogen and thawed in water at room temperature. The leaves were evaluated for cell damage with microscopic observations and wilting tests. The results provided evidence that the impregnation with trehalose by the combined actions of electric fields and vacuum impregnation drastically improved the freezing tolerance of the spinach leaves. (C) 2008 Published by Elsevier Ltd.