A formalin-inactivated Eastern equine encephalomyelitis vaccine was prepared from the PE-6 strain virus, propagated in primary chick-embryo cell cultures maintained with Medium 199 containing 0.25% human serum albumin. To effect an increase in antigenic mass per unit volume of fluid harvest, we used the pooled fluids from the first tissue-culture passage as maintenance medium for two additional passages. A formalin concentration of 1:2,000 (0.05%) at 37°C was used for inactivation of the virus suspension. Tests on the “in-process” and final vaccine documented the safety and sterility. Tests of potency on the final lyophilized product in hamsters and guinea pigs yielded ED50 values of 0.009 ml and 0.012 ml, respectively. Recent assays have revealed no change in potency after storage of the final product for 17 months at -20°C.