Abstract The preparation of diphtheria toxins from the time of its discovery by Behring has been repeatedly investigated with the development of many changes in its preparation, but it still presents many difficulties. The urgent need of strong potent toxins for the Ramon flocculation tests for titration of diphtheria antitoxins, and also for the preparation of toxin-antitoxin mixtures and toxoids for immunization, led the writer at the instigation of Dr. Park to add her attempts to the further solving of this problem. The Ramon flocculation test for titrating toxins was of greatest value in the following studies. It would have been indeed a difficult, tedious and expensive task, to check the numerous lots of toxins prepared by guinea pig instead of the test tube tests. By the Ramon tests, different lots of toxins could be tested on different days of growth and the results known from twenty minutes to twenty-four hours, depending on the strength of the toxin. Because the author was able to produce a large quantity of high grade diphtheria toxin many times in succession, her technic employed will be elaborated in detail, so that it may be of use to other workers. The details about to be outlined, except perhaps for the new factor which will be spoken about later, are nothing startling or new, except that they are coördinated, modified, simplified (no B. coli used), and also controlled by the Ramon flocculation tests and pH reactions. Since the discovery of diphtheria toxin, Roux, Yersin, Park, Williams, Theobald Smith and others; and of the newer workers—Bunker, Douglas, Hartley, Watson and Langstaff, Wilcox and others all called attention, jointly or separately, to the (1) culture employed, (2) quality of meats, (3) quantity and quality of peptones, (4) reaction of the media, (5) filtration and heat employed, (6) glassware, (7) preliminary cultures for seeding, (8) method of inoculation of large flasks for toxins, (9) length of time for production of best toxin. All these details are elaborated by the author at length.