By the use of continuous-flow zonal ultracentrifugation, a highly purified, inactivated tick-borne encephalitis (TBE) virus vaccine was prepared tht contained almost exclusively TBE virus particles. The purity of this preparation was 90- to 100-fold higher than that of a previously used partially purified vaccine. A mean protein content of 3-5 microgram/ml in the final vaccine represents 20 mouse protective doses50 in a mouse challenge potency test. This high degree of purity resulted in a strong reduction of side reactions observed with the less purified vaccine.