Alphaviruses, such as Semliki Forest virus (SFV), mature by budding at the plasma membrane (PM) of infected cells and enter uninfected ones by a membrane fusion process in the endosomes. Both processes are directed by the p62/E2-E1 membrane protein heterodimer of the virus. The p62 protein, or its mature form E2, provides a cytoplasmic protein domain for interaction with the nucleocapsid (NC) of the virus, and the E1 protein functions as a membrane fusogen. We have previously shown that the p62/E2 protein of SFV controls the membrane fusion activity of E1 through its complex formation with the latter (A. Salminen, J. M. Wahlberg, M. Lobigs, P. Liljeström, and H. Garoff, J. Cell Biol. 116:349-357, 1992). In the present work, we show that the E1 protein controls the NC-binding activity of p62/E2. We have studied E1 expression-deficient SFV variants and shown that although the p62/E2 proteins can be transported to the PM they cannot establish stable NC associations.