Summary: When physiologic salt solution was used for the extraction of mouse brains infected with St. Louis encephalitis virus, hemagglutinin appeared at varying times after the suspensions were prepared, zone phenomena, as well as incomplete patterns of agglutination, were frequently encountered, and the activity quickly disappeared on storage at 4 C, even when the large particles were removed by centrifugation at 13,000 rpm for 1 hour. When the pH of 7.2 exhibited by unbuffered saline suspensions of mouse brain was changed to 7.5 to 8.5 with dilute NaOH prior to centrifugation, the zone phenomena and incomplete patterns disappeared. It was, furthermore, found that the stability of the hemagglutinin on storage at 4 C could be maintained as long as the pH of the solution remained at about 7.7 or higher, and the larger tissue particles were removed by centrifugation. Physiologic salt solution, buffered at pH 9.0 with borate-KCl in 0.016 M concentration, was found to be an excellent fluid for the extraction of hemagglutinin from infected mouse brains. By its use, hemagglutinin of high potency was regularly demonstrated in 13,000 rpm supernates obtained immediately after preparation of the suspension. No zone phenomena were encountered and the solution could be stored in an ordinary refrigerator for at least 290 days without loss of titer. Even when this optimum method was used for extraction, no hemagglutinin could be obtained from the brains of mice infected with the Webster No. 3 strain of St. Louis encephalitis which has had a very large, although unknown, number of mouse passages over a period of 18 years.