The cytotoxicity of cadmium (Cd), arsenate (As(V)), and arsenite (As(III)) on a strain of Chlamydomonas acidophila, isolated from the Rio Tinto, an acidic environment containing high metal(l)oid concentrations, was analyzed. We used a broad array of methods to produce complementary information: cell viability and reactive oxygen species (ROS) generation measures, ultrastructural observations, transmission electron microscopy energy dispersive x-ray microanalysis (TEM-XEDS), and gene expression. This acidophilic microorganism was affected differently by the tested metal/metalloid: It showed high resistance to arsenic while Cd was the most toxic heavy metal, showing an LC 50 = 1.94 µM. Arsenite was almost four-fold more toxic (LC 50 = 10.91 mM) than arsenate (LC 50 = 41.63 mM). Assessment of ROS generation indicated that both arsenic oxidation states generate superoxide anions. Ultrastructural analysis of exposed cells revealed that stigma, chloroplast, nucleus, and mitochondria were the main toxicity targets. Intense vacuolization and accumulation of energy reserves (starch deposits and lipid droplets) were observed after treatments. Electron-dense intracellular nanoparticle-like formation appeared in two cellular locations: inside cytoplasmic vacuoles and entrapped into the capsule, around each cell. The chemical nature (Cd or As) of these intracellular deposits was confirmed by TEM-XEDS. Additionally, they also contained an unexpected high content in phosphorous, which might support an essential role of poly-phosphates in metal resistance. development of phytoplankton [5][6][7]. However, most of these studies are related to circumneutral pH water environments polluted by industrial and domestic wastes, in spite of the fact that pH has a considerable effect on the availability and, as a consequence, the toxicity of heavy metals [8].Indeed, many freshwater bodies worldwide are highly acidic (pH < 3), either due to natural causes or anthropogenic activities such as mining [9,10]. Acid drainages are due mainly to pyrite oxidation, producing significant acidity in lakes and ponds situated in areas impacted by mining, as well as in rivers receiving mine water discharge [11,12]. Additionally, extreme acidic environments tend to contain unusually high concentrations of heavy metals because their solubility increases markedly as the pH decreases [11]. Despite these extreme environmental conditions, a number of prokaryotic and eukaryotic organisms living in these extreme environments have been identified [13]. Microalgae are the dominant eukaryotes found in these ecosystems, green microalgae such as Chlamydomonas, Dunaliella, and Chlorella being the main primary producers [13].Although acidophilic algae are able to grow at high heavy metal concentrations and low pH, both of which are lethal to most eukaryotes [14], the adaptive mechanisms by which these microorganisms can survive under such environmental conditions are still poorly understood. Proteomic analysis has pointed out the importance of metal and acidity toleranc...