Summary and Conclusions In continuation of previous observations made at intervals up to 3 yr after immunization of 125 prisoner volunteers with strain E vaccine, tests have been made for the persistence of both sero-immunity and ability to resist virulent challenge at 4, 5 and 5 ½ yr after immunization. These tests are considered to represent the maximum possible extension of our observations since there remain accessible no more willing but unchallenged vaccinees. Eight vaccinated volunteers were challenged (4 at 48, 1 at 60 and 3 at 66 months) and 6 resisted, including all of the 3 tested at 66 months. The 2 exceptions were both individuals who had failed to manifest serologic response to vaccination. Assuming parallelism between ability to resist challenge infection and the presence of neutralizing antibody, it is predicted, in the light of the negligible decline in antibody level during the preceding 4 years, that the capacity to resist infection will persist for many years to come. Sera collected following challenge infection of vaccinated volunteers and normal controls were tested for CF antibody using 2 and 8 units of antigen. In confirmation of the results obtained by other workers (9, 10) it was found that use of the larger amount of antigen resulted in the demonstration of significantly higher CF reactivity in the sera from persons who had not had prior contact with the antigen and that this newly formed antibody was relatively thermolabile since inactivation at 60°C usually caused definite reduction in titers. This phenomenon also was observed when a primary antigenic stimulus was supplied by infection with avirulent strain E but was not demonstrated after non-infective Cox-type vaccine. In sera from persons who had been immunized prior to challenge, the phenomena of difference in antigen requirements and thermolability could not ordinarily be demonstrated.