The spontaneous haemolytic (SH) activity of sera was compared in groups of cultured halibut and sea bass. The optimum assay temperature was determined for each species and different red blood cell donors were tested. The effects of heat inactivation, storage temperature and of different agents like EDTA, EGTA, yeast cell components and bacterial LPS were compared. Halibut sera gave optimum lysis with sheep red blood cells (RBC) at 16 °C whereas sea bass sera showed optimum lysis with rabbit RBC at 37 °C. The haemolytic activity of halibut sera was inactivated at 45 °C while sea bass sera were inactivated at 56 °C. The haemolytic activity of halibut sera was significantly reduced during short-term storage at −80 °C, whereas the sea bass sera maintained fairly good activity after 1-year storage at −80 °C. EGTA and EDTA inhibited the spontaneous haemolytic activity of sera from both the species. Zymosan and MacroGard from yeast cells also inhibited the haemolytic activity of the sera of both species, whereas LPS had a very slight effect. Considerable variation in haemolytic activity was observed within both the halibut and sea bass groups studied.