Technical Abstract: The need for a non-thermal intervention technology that can achieve microbial safety without altering nutritional quality of liquid foods led to the development of the radio frequency electric fields (RFEF) process. However, insight into the mechanism of bacterial inactivation by this technology is limited. Apple juice purchased from a wholesale distributor was inoculated with Escherichia coli K-12 at 7.8 log CFU/ml and then treated with RFEF. The inoculated apple juice was passed through an RFEF chamber set at 25 kV/cm and operated at 25 deg C, 55 deg C and 75 deg C for 3.4 milliseconds at a flow rate of 540 ml/min. Treated samples were monitored for changes in cell membrane using Transmission Electron Microscopy (TEM), bacterial cell surface hydrophobicity and charge using electrostatic and hydrophobic interaction chromatography. Surviving cell populations including injured cells was determined by plating 0.1 ml sample on Tryptic Soy Agar and Violet Red Bile Agar, with 5 ml overlay of the same agar containing 4-methylunbelliferyl-beta-D-glucuronide. RFEF treatment at 55 deg C caused a significant decrease in the surviving populations of E. coli cells compared to heat treatment alone at 75 deg C for 5 min. RFEF treatment caused a significant decrease in surface hydrophobicity and negative ion charge of E. coli cells. TEM observation of treated samples showed variation in internal membrane damage of the bacteria. The results of this study suggest that the mechanism of inactivation of RFEF is by disruption of the bacterial cell surface hydrophobicity and loss of relative negative ions which led to changes in the internal structure of E. coli membrane and death.