The structure of the intracellular form of φX174 DNA was studied in cells treated to inhibit protein synthesis before infection. Inhibition of protein synthesis was accomplished in Escherichia coli 15 THU by 90 minutes incubation in medium lacking histidine and uracil, and in E. coli THU or HF4704 by addition of 135 μg chloramphenicol/ml. five minutes before infection. φXDNA containing parental label was isolated from treated cells. Its sedimentation rate in a neutral high salt gradient was 5% faster than RFI † † Abbreviations used: RF, replicating form; BNC, benzoyl naphthoyl DEAE-cellulose. , and the buoyant density in neutral cesium chloride was more broadly distributed and somewhat denser than RFI. When the intracellular parental labeled DNA was fractionated by chromatography on benzoyl naphthoyl DEAE-cellulose 10 to 20% chromatographed as RFI or II, while 80 to 90% was tightly bound and could only be eluted by 1.0 m-NaCl plus 2% caffeine. The tightly bound DNA was infective in the spheroplast assay, but infectivity was lost after denaturation. In alkaline CsCl equilibrium gradients the parental label was found in the position of viral DNA but no infectivity was found in the region of viral or complementary strand. Hybrid ( 13C, 15N parental: 12C, 14N post-infection) DNA had a lower buoyant density than normal hybrid RF. Parental strands sedimented slower than viral DNA on alkaline CsCl gradients with a peak around ~ 8 s. Post-infection DNA had a heterogeneous sedimentation distribution in alkali, but a majority of the counts sedimented faster than viral DNA. Most of the parental label was membrane associated, and all infected cells were able to contribute to initial progeny phage production. The structural and biological significance of these findings is discussed.