The ciliated protozoon Euplotes focardii, originally isolated from the coastal seawaters of Terra Nova Bay in Antarctica, shows a strictly psychrophilic phenotype, including optimal survival and multiplication rates at 4e5 C. This characteristic makes E. focardii an ideal model species for identifying the molecular bases of cold adaptation in psychrophilic organisms, as well as a suitable source of novel cold-active enzymes for industrial applications. In the current study, we characterized the patatin-like phospholipase from E. focardii (EfPLP), and its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcPLP). Both EfPLP and EcPLP have consensus motifs conserved in other patatin-like phospholipases. By analyzing both esterase and phospholipase A2 activity, we determined the thermostability and the optimal pH, temperature dependence and substrates of these enzymes. We demonstrated that EfPLP shows the characteristics of a psychrophilic phospholipase. Furthermore, we analyzed the enzymatic activity of three engineered versions of the EfPLP, in which unique residues of EfPLP, Gly80, Ala201 and Val204, were substituted through site-directed mutagenesis with residues found in the E. crassus homolog (Glu, Pro and Ile, respectively). Additionally, three corresponding mutants of EcPLP were also generated and characterized. These analyses showed that the substitution of amino acids with rigid and bulky charged/hydrophobic side chain in the psychrophilic EfPLP confers enzymatic properties similar to those of the mesophilic patatin-like phospholipase, and vice versa. This is the first report on the isolation and characterization of a cold-adapted patatin-like phospholipase from eukaryotes. The results reported in this paper support the idea that enzyme thermaladaptation is based mainly on some amino acid residues that influence the structural flexibility of polypeptides and that EfPLP is an attractive biocatalyst for industrial processes at low temperatures.