Cryo-electron microscopy three-dimensional reconstructions have been made of mature and of emptied bacteriophage ϕ29 particles without making symmetry assumptions. Comparisons of these structures with each other and with the ϕ29 prohead indicate how conformational changes might initiate successive steps of assembly and infection. The 12 adsorption capable ‘appendages’ were found to have a structure homologous to the bacteriophage P22 tailspikes. Two of the appendages are extended radially outwards, away from the long axis of the virus, whereas the others are around and parallel to the phage axis. The appendage orientations are correlated with the symmetry-mismatched positions of the five-fold related head fibers, suggesting a mechanism for partial cell wall digestion upon rotation of the head about the tail when initiating infection. The narrow end of the head-tail connector is expanded in the mature virus. Gene product 3, bound to the 5′ ends of the genome, appears to be positioned within the expanded connector, which may potentiate the release of DNA-packaging machine components, creating a binding site for attachment of the tail.