Possible non-thermal effects of radio frequency electromagnetic fields (RF-EMF) on retinal ganglion cells were studied in vitro under conditions of constant temperature. Isolated mouse retinae were exposed to GSM-900, GSM-1800, and universal mobile telecommunication system (UMTS) RF-EMF applying specific absorption rates (SAR) of 0 (sham), 0.02, 0.2, 2, and 20 W/kg. Temperature was kept constant within ±0.5 to 1 °C for GSM-900 and ±0.5 °C for GSM-1800 and UMTS. Responses of retinal ganglion cells to light stimuli of three intensities (0.5, 16, and 445 lx) were recorded before, during, and up to 35 min after exposure. Experiments were performed under double-blind conditions. Changes in light responses during and after exposure were determined for each condition (RF-EMF; SAR value; light intensity) with respect to the responses before exposure, respectively. Changes were calculated using the Euclidian distance of the n-dimensional response vectors, respectively. Some changes already occurred during sham (0 W/kg) exposure, reflecting the intrinsic variability in retinal ganglion cell responses. Comparison of the distance values from sham exposure with those from actual exposure yielded no significant differences. In addition, linear regression analysis of the distance values versus SAR values yielded no consistent dependence of light response changes. From these results we conclude that RF-EMF exposure at three mobile phone frequencies (GSM-900, GSM-1800, UMTS) and SARs up to 20 W/kg has no acute effects on retinal ganglion cell responses under constant temperature conditions.