In this study we investigated the use of a DNA dosimeter to accurately measure changes in ultraviolet B radiation (UVBR; 280–315 nm) under Antarctic ozone hole conditions. Naked DNA solution in quartz tubes was exposed to ambient solar radiation at Rothera Research Station, Antarctica, between October and December 1998 for 3 h during UVBR peak hours (1200–1500 h). Trends in UVBR-mediated DNA damage (formation of cyclobutane pyrimidine dimers [CPD]) were related to cloud cover, ozone-column depth and spectroradiometric measurements of ambient radiation. Ozone-column depths ranged from 130 to 375 DU during the study period, resulting in highly variable UVBR doses, from 1.6 to 137 kJ m⁻² over the 3 h exposure, as measured by spectroradiometry. There was a strong positive correlation (86%) between dosimeter CPD concentrations and DNA-weighted UVBR doses. Ozone depth was a strong predictor of DNA damage (63%), and there was no significant relationship between CPD formation and cloud cover. Subtle changes in spectral characteristics caused by ozone depletion were detected by the biodosimeter; the highest CPD concentrations were observed in October when ozone-mediated shifts favored shorter wavelengths of UVBR. We conclude that the DNA biodosimeter is an accurate indicator of biologically effective UVBR, even under highly variable ozone conditions.