A preparative method is described for the isolation of the major protein species from the virion of bacteriophage phiX174. Two proteins, the cistron G and H products, are located in the virion spikes. After removal of the spikes, the capsid contains the cistron F product as well as a small protein which is the product of cistron J and the majority of the DNA. During the removal of the spikes, a precipitate containing the F and G proteins is formed. The proteins from the spike, capsid, or precipitate can be isolated on the basis of size by gel-filtration chromatography. The cistron G protein has an aminoterminal methionine, while the small J protein has an amino-terminal serine. Amino acid compositions as well as peptide maps indicate each species is unique and that, in sum, they account for over half the coding capacity of the viral genome.