Phagocytosis is a fundamental defense mechanism shared by both invertebrates and vertebrates, mediated by specific cells (phagocytes) rich in lysosomal granules. As tunicates seem to occupy a key position in the evolution of vertebrates, we decided to study phagocytosis in the colonial ascidian Botryllus schlosseri, as a subphylum representative, in order to provide some more data on this defense strategy in protochordates. In vitro phagocytosis in Botryllus is sensitive to incubation temperature and test particle concentration. It is dependent on the presence of opsonising factors and, in the case of yeast, is mediated by a direct interaction between phagocyte membrane and yeast surface through a mannose receptor, namely a calcium-dependent membrane lectin with specificity for mannose and its derivatives. Humoral factors affecting phagocytosis are present in the blood plasma (BP) as ingestion of living yeast and sheep red blood cells (SRBC) is significantly reduced after preincubation of these particles with whole BP. In these experimental conditions, the formation of test particle aggregates suggests the presence in BP of an agglutinin able to coat yeast and SRBC thus limiting their availability to phagocytes. The effect disappears after autoclaving of yeast or formalinization of SRBC, probably because of a modification of cell surface molecules during the treatment. Preincubation of the above test particles either with yeast- or SRBC-absorbed BP or with subagglutinating concentrations of BP reveals the presence of an opsonin able to enhance phagocytosis. Simultaneously with the process of particle ingestion, extracellular degranulation of the lytic content of phagocyte granules occurs in vertebrates. In Botryllus we have demonstrated an increase in exocytosis of hydrolases after incubation of hemocytes with test particles. The production of reactive oxygen intermediates (ROI) during phagocytosis is well known for vertebrates and has been recently reported in several invertebrates: it is consequent to an increase of oxygen consumption (respiratory burst) in activated phagocytes. Our study on B. schlosseri shows that ROI are produced during ascidian phagocytosis: in both cytochemical and spectrophotometric assays, we have measured a significant increase in the production of superoxide anion and hydrogen peroxide after only 5 min of incubation of hemocytes with the test particles. A significant increase in reactive nitrogen intermediates (RNI) secretion, as increase in extracellular nitrite ions, was also observed after 30 min of incubation with yeast cells. Fixed heterologous hemocytes form nonfusible colonies are phagocytised at a significant higher extent than autologous fixed hemocytes thus revealing the capacity of allorecognition in phagocytes. Three cell types are involved in phagocytosis, namely hyaline amoebocytes, macrophage-like cells and signet-ring cells. Hyaline amoebocytes represent the first cell type showing ingested particles after few minutes of incubation whereas macrophage-like and signet-ring cells are the prevailing phagosome containing cell types after 30 min of incubation. This result supports our hypothesis of a close relationship between the three hemocyte types, hyaline amoebocytes being the probable precursors of macrophage-like and signet-ring cells.