Whether or not it is related to stratospheric ozone depletion, Antarctic microalgae experience ultraviolet-B radiation (UVBR) stress in situ, leading to decreased photosynthetic performance, DNA damage, and/or altered community composition. UVBR vulnerability is known to be species specific, but may also be affected by a range of environmental growth conditions, including the light history of the cells. This study investigates the influence of acclimation to photosynthetically active radiation (PAR) on the vulnerability to UVBR-induced DNA damage in Antarctic microalgae. Chaetoceros dichaeta, Pyramimonas gelidicola, Phaeocystis antarctica and Polarella glacialis were acclimated to 5 PAR levels, after which growth rate, pigment composition, malondialdehyde (MDA, a general indicator of oxidative stress) and UV-absorbing compounds were measured. Photoacclimated cultures were then exposed to a single UVBR treatment and the accumulation of UVBR-induced DNA damage was determined by the number of cyclobutane pyrimidine dimers (CPDs). Acclimation to increasing irradiance enhanced both the xanthophyll to chlorophyll a ratio and xanthophyll deepoxidation in all species. Increased cellular MDA levels were found at the highest irradiance in all species except P. gelidicola. Yet, growth rates were only reduced at the 2 lowest PAR levels. P. antarctica and P. glacialis showed a strong linear induction of UV-absorbing compounds at increasing PAR intensities, whereas P. gelidicola showed no induction of these compounds. The UVBR treatment induced CPDs in P. gelidicola only, and CPD levels were elevated at the highest PAR acclimation intensities. Thus, sensitivity to UVB-induced CPD accumulation was species specific, and, counterintuitively, acclimation to high PAR increased the sensitivity of P. gelidicola to UVB-induced DNA damage.