Some properties of φA, the viral DNA and the intracellular RF were investigated. The virus particle is spherical and its diameter is about 300 AÅ. φA is immunologically related to φX174 and physical properties such as optical rotatory dispersion were similar to those of φX174. The plating efficiency of φA on several E. coli strains was compared with those of other phages of φX174 group. The infectivity of the virion DNA is extremely sensitive to hydroxylamine treatment, UV* irradiation or DNase I [EC 3.1.4.5], but is refractory to exonuclease I of E. coli. Spheroplast infectivity of φA DNA is inhibited by heterologous denatured DNA but not by native double-stranded DNA. Like φX174 DNA, φA DNA shows hyperchromicity upon treatment with formaldehyde. Its behaviour in methylated-albumin column chromatography or sucrose-density-gradient centrifugation of φA RF closely resembled that of φX174. The infectivity of φA RF increases several-fold on alkaline treatment, but is unaffected by incubation with hydroxylamine. Although φA RF is sensitive to DNase I, the inactivation rate is low as compared with the virion DNA. Survival of UV-irradiated RF is remarkably influenced by the uvr function of host cells and, in hcr+ bacteria, the UV sensitivity of RF is far lower than that of the virion DNA. The infectivity of φA RF to lysozyme-EDTA spheroplasts was inhibited specifically by double-stranded heterologous DNA, and infectivity of φA DNA to Ca2+-treated cells was inhibited by denatured DNA. These results show that while φA DNA is single-stranded, the RF molecule is double-stranded.