Inactivation of Semliki Forest virus (SFV) with formalin, β-propiolactone, hydroxylamine and 2-ethyl ethylenimine was studied. Immunogenicity of SFV was best retained after formalin inactivation. Vaccines were pre pared by applying two inactivation procedures in the following order: (a) disruption of SFV by either Tween 80- ether, NP 40 or deoxycholate, or treatment of SFV with saponin; (b) addition of either formalin, β-propiolactone, hydroxylamine or 2-ethylethylenimine. These vaccines were evaluated in white mice, in several experiments. It was concluded that a gradient from potent to less- or non-potent vaccines exists in the order saponin-formalin, Tween 80-ether-formalin and NP 40-ethylethylenimine or deoxycholate-formalin inactivation, but none of these vaccines was superior to reference vaccines containing only formalin-inactivated viru