The lytic cycle of Bacillus subtilis phage 41c required the presence of at least 10 mM-calcium. In the absence of this ion, the plaquing efficiency of the virus was reduced to less than 0.1. Likewise, replacement of Ca2+ with other divalent ions (Ba2+, Sr2+, Mg2+, Mn2+) resulted in reduced efficiencies. Adsorption of 41c was Ca2+-dependent, requiring concentrations ranging from 0.1 to 10mM. Although more than 90% of the phage adsorbed at 0.1 mM-Ca2+, successful infection could only be achieved at higher Ca2+ levels. Sub-optimal concentrations of the ion resulted in the loss of 90% of infected centres within 1 min after the initiation of infection, indicating an early post-adsorption ion requirement. Penetration of experiments with 32P-labelled phage DNA indicated than an irreversible inhibition of injection was occurring in the majority of the phage-bacterium complexes. A third level of cation involvement became apparent when phage-bacterium complexes in which penetration had occurred exhibited a greatly reduced burst size. The post-penetration ionic requirement occurred early in the infection process since dilution of infected complexes into Ca2+-free medium at 2.5 min p.i. resulted in reduced phage yields. The requirement was dispensable after 6 min p.i., since infected complexes diluted into Ca2+-free medium at this time exhibited a normal one-step growth curve. Analysis of messenger RNA production by molecular DNA-RNA hybridization techniques indicated that transcriptional events were similar in the presence and absence of Ca2+. At present, the identification of the third ion-dependent stage is unresolved.