Objective An electronegative electroretinogram (ERG) can indicate important ocular or systemic disease. This study explored the prevalence of electronegative responses to dark-adapted stimuli in a largely healthy cohort. Methods and Analysis 211 participants recruited from the TwinsUK cohort underwent ERG testing incorporating international standard (International Society for Clinical Electrophysiology of Vision (ISCEV)) protocols and additional stimuli. Responses were recorded using conductive fibre electrodes, following pupil dilation and 20 min dark adaptation. Responses analysed were to the ISCEV standard and strong flashes (3.0 and 10 cd/m 2 s), and to additional white flashes (0.67-67 cd/m 2 s). A-wave and b-wave amplitudes were extracted; b:a ratios were calculated and proportions of eyes with ratios<1 were noted. Results Mean (SD) age was 62.4 (11.4) years (median, 64.3; range 23-86 years). 93% were female. Mean (SD) b:a ratios for right and left eyes, respectively, were 1.86 (0.33) and 1.81 (0.29) for the standard flash, and 1.62 (0.25) and 1.58 (0.23) for the stronger flash; average b:a ratio was lower for the stronger flash (p<0.0001). No waveforms were electronegative. For additional flashes, b:a ratio decreased with increasing flash strength. No electronegative waveforms were seen except in three eyes (0.7%) for the strongest flash; in some cases, drift in the waveform may have artefactually reduced the b:a ratio. Conclusion For standard dark-adapted stimuli, no participants had electronegative waveforms. The findings support the notion that electronegative waveforms (in response to standard flash strengths) are unusual, and should prompt further investigation.