This study aimed to evaluate the use of nalidixic acid-adapted strains of three major Shiga toxin-producing Escherichia coli (STEC) and non-pathogenic E. coli for the use of radio frequency (RF) heating using phosphate buffer saline (PBS). The effectiveness of RF was evaluated on cocktails of various STEC serotypes (O157:H7, O26:H11, O111) and non-pathogenic E. coli at different endpoint temperatures (55, 60, and 65 °C). All strains were successfully adapted to nalidixic acid (Nal). In general, the results indicated that Nal-adapted strains were not significantly different from Nal-sensitive strains evaluated either before treatment or at the endpoint temperatures. Nal-adapted strains were, therefore, confirmed to be effective as marker organisms in studies involving the use of RF in buffer. Results also showed that the thermal inactivation of strains was more effective as the treatment temperature increased, particularly at 65 °C, which showed a 6.0 log CFU/ml reduction. The results of the present study serve as a baseline to study RF as a potential intervention technology for non-intact beef products. [ABSTRACT FROM AUTHOR]