Semliki Forest virus (SFV) is an enveloped animal virus comprising an icosahedral nucleocapsid surrounded by a membrane containing 80 transmembrane, trimeric spikes. SFV was treated with the non-ionic detergent n-octyl beta-D-glucopyranoside (octylglucoside) and analysed by cryo-electron microscopy and image reconstruction to explore the interaction between the spikes and the capsid. Comparison of the structure of detergent treated SFV (DSFV) with SFV by three-dimensional image reconstruction from cryoelectron micrographs showed that one fourth of the spikes, those on the 3-fold axis, were selectively removed by detergent treatment. Quantitative immunoblotting of gently detergent treated virus showed that polypeptide E1 was selectively removed from the trimeric spike complex (E1, E2, E3)3. Difference imaging between DSFV and SFV in combination with comparison to the previously established structure of Sindbis virus, which lacks the E3 protein, leads to a model for the position of E1, E2 and E3 in the spike. If the trimeric spike is represented as a triangle, E2 extends from the centre to the vertices and E1 fills in between the ridges of E2 to form the edges of the triangle while E3 is at the distal end of the spike, interacting primarily with E2.