In a first phase of this investigation, a validation of our elevated plus-maze apparatus was performed in male Sprague–Dawley rats by testing anxiety response at various ambient light intensities (200, 30, 10 and 2.5 lux), as well as the effects of diazepam treatment (0.5 and 1.0 mg/kg, i.p. at 30 lux). Anxiety responses were found to decrease with decreasing light intensity and to be attenuated by diazepam treatment. Subsequently, a separate set of rats was exposed to 2.45 GHz EMFs (2 μs pulse width, 500 pulses per second, whole-body and time averaged of SAR 0.6 W/kg ±2 dB, brain-averaged SAR of 0.9 W/kg ±3 dB) for 45 min to assess whether EMF exposure altered anxiety responses in the same apparatus. As we made no a priori hypothesis on whether the effects would be anxiogenic or anxiolytic, part of the rats were tested under an ambient light intensity of 2.5 lux, the other one being tested at 30 lux. The low intensity level set the behavioural baseline for the detection of anxiogenic effects, while the higher one corresponded to the detection of anxiolytic effects. Sham-exposed and naive rats were used as controls. Whatever light intensity was used, EMF exposure failed to induce any significant effect on anxiety responses in the plus maze. The present experiment demonstrates that exposure to EMFs, which was previously found to increase the number of benzodiazepine receptors in the rat cortex [Lai H, Carino MA, Horita A, Guy AW. Single vs. repeated microwave exposure: effects on benzodiazepine receptors in the brain of the rat. Bioelectromagnetics 1992;13(1):57–66], does not alter anxiety responses assessed in the elevated plus maze.