Enterococci are the causative agents of several serious infections. In addition, antibiotic- resistant enterococci are becoming increasingly prevalent due to the extensive use of antibiotics in the community and in hospitals. In this context, new antimicrobials acting by different mechanisms are highly desired. Lysins are bacteriophage-derived molecules that are produced when the bacteriophage (or phage) progeny needs to escape the bacterial host. Lysins very rapidly lyse the bacterial peptidoglycan. They can reach their target when added exogenously to Gram-positive bacteria, thanks to the lack of an impermeable outer membrane. In contrast, they are inactive when added externally to Gram-negative bacteria, which are surrounded by bilayer outer membrane. The action of lysins is normally restricted to bacterial hosts of the phages from which they were isolated, but some lysins have a broader spectrum of activity. In the present work, new lysins with a potentially lytic action against enterococci were identified by consulting a database of prophage sequences within bacterial genomes. Their amino acid sequences were analyzed in order to highlight both functional domains and possible differences with the previously described lysins. Three new lysins (plyEF, plyHH22 and plyE613) and the previously described enterococcal lysin plyV12 were studied. Cloning and purification were performed using several bacterial expression vectors with and without a C-terminal six-histidine tag. The expression and activity of lysins were screened by different methods. Recombinant host bacteria (E. coli BL21) were grown in various media and lysin expression was induced with IPTG. Induction of plyEF, plyHH22 and plyE613 readily blocked the growth of E. coli recipients, suggesting that they were toxic for this particular host. Thus, these enzymes could not be further purified. In contrast, induction of plyV12 did not block E. coli growth and the enzyme could be further studied. The latter results confirm the validity of our protocol. Moreover, we could also confirm the rapid lytic activity of plyV12 against E. faecalis ATCC 29212. The reason of the toxicity of plyEF, plyHH22 and plyE613 toward E. coli is as yet unclear and needs to be further explored.