Monoclinic P2(1) crystals of the bacteriophage phi X174 have been incubated with calcium ions (Ca2+) and the induced structural conformational changes studied to 3 A resolution with X-ray crystallographic methods. Three different types of Ca2+ binding sites have been located within the asymmetric unit of the virion. Two sets of sites are associated with the F capsid protein. One set of sites associated with the F protein is in a general position near the icosahedral 3-fold axes of the virus, with the main-chain carbonyl oxygen atoms of residues Gly1321, Asp1421, Met1424 and Ser1426, and the side-chains of Gln1004 and Asp1421 as ligands. The other set of sites associated with the F protein is on the icosahedral 3-fold axes, with the symmetry-related main-chain carbonyl oxygen atoms of Ser1001 and the side-chains of Asn1002 as ligands. The bound Ca2+ induce a conformational change of the amino-terminal residues of the F proteins. A third set of sites, consisting of a pair of Ca2+ on the icosahedral 5-fold axes, are associated with the G spike protein and are concurrently liganded by the symmetry-related carbonyl oxygen side-chains of Asp2117. Concomitant with the binding of Ca2+ to the phage is the rotation of the Asp1209 side-chain of the F protein towards some additional electron density that was not observed in the absence of Ca2+. This density is situated in a shallow depression near the icosahedral 2-fold axes of the virus, and has been tentatively interpreted as a bound glucose molecule that is ordered only in the presence of Ca2+. The putative glucose binding site may be related to the attachment of the virus to cell surface lipopolysaccharides in the initial stages of Escherichia coli infection.