Photoprotection against UV-B radiation (UVBR; 280–320 nm) was examined in natural phytoplankton communities from two coastal environments at different latitudes: temperate Rimouski (Canada) and tropical Ubatuba (Brazil). Mesocosm experiments were performed at these sites to examine the response of phytoplankton to increases in UVBR that corresponded to local depletions of 30% and 60% in atmospheric ozone levels (low and high UVBR treatments, respectively). A fluorescence method using a pulse amplitude modulation fluorometer (Xe-PAM, Walz, Germany) with selective UV filters was used to estimate photoprotection, and these results were compared with an index of mycosporine-like amino acid (MAA) concentrations determined using spectrophotometry of methanol extracts. The present study provided the first evidence, to our knowledge, of the suitability of this in vivo fluorescence method for the estimation of UV photoprotection efficiency in natural phytoplankton. No significant differences were found for most of the variables analyzed between the light treatments used at both sites, but differences were found between sites throughout the duration of the experiments. Vertical mixing, used to maintain cells in suspension, likely alleviated serious UVBR–induced damage during both experiments by reducing the length of time of exposure to the highest UVBR irradiances at the surface. In Rimouski, this was the main factor minimizing the effects of treatment, because optical properties of the coastal seawater rapidly attenuated UVBR throughout the water column of the ca 2 m deep mesocosms. In this location, synthesis of MAAs and photoprotective pigments likely contributed to the observed phototolerance of phytoplankton and, hence, to their growth; however, in a comparison of the UVBR treatments, these variables showed no differences. In Ubatuba, where nutrient concentrations were significantly lower than those in Rimouski, light attenuation was less than that in Rimouski and UVBR reached the bottom of the mesocosms. UVBR penetration and the forced vertical mixing of the cells, without the possibility of vertical migration below this photostress zone, resulted in photo-inhibition, because confinement in the mesocosms forced cells to remain constantly exposed to high levels of irradiance during the daytime. Hence, additional effects of UVBR were masked in this experiment, because cells were damaged too much and phytoplankton populations were rapidly declining. There was also an overall preservation of MAAs, in contrast with chlorophyll (Chl) degradation, in spite of the fact that this UV screening was not sufficient to counteract photo-inhibition, which suggests an important role for these molecules, either in the overall photoprotection strategy or in other physiological processes. Altogether, local water characteristics, namely attenuation, mixing, and nutrients concentration, can strongly modulate the photoprotection strategies used by natural phytoplankton populations in coastal environments.