The Semliki Forest virus (SFV) spike is composed of three copies of a membrane protein heterodimer. The two subunits of this heterodimer (p62 and E1) are synthesized sequentially from a common mRNA together with the capsid (C) in the order C-p62-E1. In this work heterodimerization of the spike proteins has been studied in BHK 21 cells. The results indicate that: (a) the polyprotein is cotranslationally cleaved into individual chains; (b) the two membrane protein subunits are initially not associated with each other in the endoplasmic reticulum (ER); (c) heterodimerization occurs predominantly between subunits that originate from the same translation product (heterodimerization in cis); (d) the kinetics of subunit association are very fast (t1/2 = 4 min); and (e) this heterodimerization is highly efficient. To explain the cis-directed heterodimerization reaction we suggest that the p62 protein, which is made before E1 during 26S mRNA translation, is retained at its translocation site until also the E1 chain has been synthesized and translocated at this same site. The mechanism for p62 retention could either be that the p62 anchor sequence cannot diffuse out from an "active" translocation site or that the p62 protein is complexed with a protein folding facilitating machinery that is physically linked to the translocation apparatus.