A protoplasmic drop isolated from an internodal cell of Nitella became electrically excitable in response to an external stimulus when the drop was placed in a test solution containing Ca 2+, Mg 2+ and other appropriate ions. The structure of the surface membrane of the protoplasmic drop was determined by measuring the refractive index and the electrical properties of the membrane with and without enzymes in the external solution. The results obtained are summarized as follows. 1. 1. The refractive index of the membrane, determined from the measurements of the Brewster angle, decreased with time from 1.47 ± 0.02 to 1.42 ± 0.02 within about 1 h after a drop was formed in the test solution. 2. 2. The membrane potential decreased gradually with time and approached a steady value of about −90 mV or −50 mV, while the membrane resistance rose temporarily and then decreased and approached a steady value between 0.4 and 3 kΩ·cm 2 within about 1 h. The membrane became electrically excitable when the membrane potential and the resistance attained the study values mentioned above. 3. 3. The presence of proteases, e.g. trypsin and pronase, in the external medium during the formative period of the excitable membrane inhibited the decrease of the membrane potential and of the membrane resistance. The excitability of the membrane was not induced in this situation. Removal of the proteases from the external solution, however, led to the formation of the excitable membrane. 4. 4. After the membrane became excitable, an application of proteases led to a depolarization of the membrane potential and an increase of the membrane resistance, and the excitability was suppressed. 5. 5. Addition of phospholipase A, 0.05 mg/ml, caused an immediate destruction of the drop at any stage of membrane formation. These results imply that the surface membrane of a protoplasmic drop is formed initially from phospholipids. Then proteins penetrate partially through the surface membrane and combine with the phospholipids in the membrane, leading to an appropriate conformation of the membrane macromolecules responsible for the excitability of the protoplasmic drop.