Phenoloxidases (POs) constitute a family of copper-containing enzymes with orthodiphenoloxidase (catecholase) activity widely distributed among invertebrates. They exert a pivotal role in immune defences as they can induce cytotoxicity through the conversion of phenols to species. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules as well as in the formation of the cytotoxic foci along the contacting edges of genetically incompatible colonies which characterises the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been identified in C. intestinalis (Immesberger and Burmester, 2004). In the present study, we determined the cDNA sequences of the POs from two colonial ascidians: Botryllus schlosseri from Mediterranean (Adriatic) Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments clearly evidenced the similarity between ascidian PO and crustacean proPOs whereas the analysis of the three-dimensional structure of compound ascidian POs reveal high similarity with arthropod haemocyanins which share common precursors with proPOs. Ascidian POs and arthropod proPOs grouped in the same cluster well separated from mollusc tyrosinases, and share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanins, involved in the regulation of enzyme activity. Cytoenzymatic studies and in situ hybridisation (ISH) indicated that the genes are transcribed inside morula cells (MCs), a characteristic haemocyte type in ascidians, at the beginning of their differentiation. Sequence analysis allowed a better understanding of previous biochemical data and suggest some hypothesis for the regulation of enzyme activity.