Sindbis virus contains two membrane glycoproteins, E1 and E2, which are organized into 80 trimers of heterodimers (spikes). These trimers form a precise T=4 icosahedral protein lattice on the surface of the virus. Very little is known about the organization of the E1 and E2 glycoproteins within the spike trimer. To gain a better understanding of how the proteins E1 and E2 are arranged in the virus membrane, we have used the techniques of limited proteolysis and amino acid chemical modification in combination with mass spectrometry. We have determined that at neutral pH the E1 protein regions that are accessible to proteases include domains 1-21 (region encompassing amino acids 1 to 21), 161-176, and 212-220, while the E2 regions that are accessible include domains 31-84, 134-148, 158-186, 231-260, 299-314, and 324-337. When Sindbis virus is exposed to low pH, E2 amino acid domains 99-102 and 262-309 became exposed while other domains became inaccessible. Many new E1 regions became accessible after exposure to low pH, including region 86-91, which is in the putative fusion domain of E1 of Semliki Forest virus (SFV) (M. C. Kielian et al., J. Cell Biol. 134:863-872, 1996). E1 273-287 and region 145-158 were also exposed at low pH. These data support a model for the structure of the alphavirus spike in which the E1 glycoproteins are centrally located as trimers which are surrounded and protected by the E2 glycoprotein. These data improve our understanding of the structure of the virus membrane and have implications for understanding the protein conformational changes which accompany the process of virus-cell membrane fusion.