When, however, such eggs were given a short treatment with hypertonic sea water or with lack of oxygen or with KCN they developed into normal larvæ. This new or improved method of artificial parthenogenesis is as follows: The eggs are put for from two to four minutes into 50 c.c. sea water containing a certain amount of N/10 butyric acid (2.6 c.c. in the case of S. _purpuratus_ in California and 2.0 c.c. in the case of _Arbacia_ in Woods Hole). Ten or fifteen minutes later the eggs are put into hypertonic sea water (50 c.c. sea water+8 c.c. 2-1/2 m NaCl or Ringer solution or cane sugar) in which they remain, at 15° C. from thirty-five to sixty minutes in the case of _purpuratus_, and from 17-1/2 minutes to 22-1/2 minutes at 23° in the case of _Arbacia_ at Woods Hole. If the eggs are then transferred to normal sea water they will develop. In making these experiments, which have been repeated and confirmed by numerous investigators, it should be remembered that this effect of the hypertonic solution has a high temperature coefficient (about two for 10° C.) and that a slight overexposure to the hypertonic sea water injures the eggs so that development is abnormal. By this method it is possible to imitate the activating effect of the living spermatozoön upon the egg in every detail and eggs treated in this way will develop in large numbers into perfectly normal larvæ. We shall see later that they can also be raised to the adult state. 2. The next task was to find out the nature of the action of the two agencies upon the development of the egg. It soon became obvious that the membrane formation (or the alteration underlying membrane formation) was the more important of the two, since in the eggs of starfish and annelids this was sufficient for the production of larvæ, and that the second treatment had only the corrective effect, of overcoming the sickly condition in which mere membrane formation had left the eggs. It was, therefore, of great interest to ascertain what substances or agencies caused membrane formation in the egg, since it now became clear that the spermatozoön could only cause membrane formation by carrying one such substance into the egg. These investigations led the writer to the result that all those substances and agencies which are known to cause cytolysis or hemolysis (see Chapter III) will also induce membrane formation, and that the essential feature in the causation of development is a cytolysis of the superficial or cortical layer of the egg. As soon as this layer is destroyed the development of the egg can begin.