Abstract: A method for producing horseshoe crab amebocytes in vitro by isolating amebocyte producing tissue from the gill flaps of horseshoe crabs, especially Limulus polyphemus. In contrast to all previous attempts to isolate and culture amebocytes from Limulus for the production of pyrogen-sensitive lysate, an effective method and means has been developed utilizing the discovery that the gill flaps are the source of the cells which differentiate into competent amebocytes. Once isolated, the tissue can be cultured long term in vitro on an artificial surface or as part of the gill flap leaflets, opened along one edge to allow access of the media to the developing amebocytes. Amebocytes are removed from the gill flaps by pulsing with Limulus serum, copper sulfate, detergent, or combinations thereof.