X-ray diffraction data were collected from crystalline southern bean mosaic virus soaked in EDTA at pH 5.5. Under these conditions, the virus does not swell, although much of the Ca 2+ and Mg 2+ is removed from the virions. A difference electron density map, at 5.0-Å resolution, between this and the native data showed three independent peaks per icosahedral asymmetric unit. One was located on the quasi-threefold axis at a site where a cation would be liganded to three quasi-related glutamates. The other two were located on the protein-RNA interface related by the quasi-threefold axis, thus leaving the other related site vacant. Atomic absorption spectroscopy results suggest that these sites are all occupied by calcium ions.