Mercedes Hernando-Pérez, Roberto Miranda, María Aznar, José L. Carrascosa, Iwan AT Schaap, David Reguera, and Pedro J. de Pablo* experimental evidence for the pressurization of DNA inside phages. In particular, a variety of experiments have shown that by imposing an external osmotic pressure using osmolites, it is possible to counteract this internal pressure and to control the DNA ejection process in the phages λ [8] and T5.[9] Furthermore, both theoretical [10, 11] and experimental [12, 13] results indicate that the packaging processes of the DNA inside phages (Figure 1 a2) requires forces about 50–100 pN. These forces can be translated into estimates of the pressurization of the genome inside the capsid, resulting in 50 to 100 atm, although these techniques do not offer a direct measurement of the phage internal pressure. Here we provide evidence of internal pressure in phage ϕ 29 by using atomic force microscopy (AFM) nanoindentation experiments which directly probe the mechanical stiffness of individual viral particles. By comparing the effective spring constant (elasticity) of DNA full virions and DNA-devoid particles, we have been able to measure the contribution of the confined DNA to the stiffness of the shell and the subsequent evaluation of its internal pressure. Moreover, we also show that this pressure can be reversibly modified by the presence of counterions that reduce the electrostatic repulsion of the confined DNA. Double-stranded DNA bacteriophages first assemble in an icosahedral prohead (Figure 1 a1), which is later filled with DNA during the maturation process (Figure 1 a2). Bacteriophage ϕ 29 (see Supporting Information (SI)), is constructed from 235 gp8 subunits arranged in 11 pentameric plus 20 hexameric units forming icosahedral end caps, and 10 hexameric units forming the cylindrical equatorial region.[14] In one of the end caps, the central pentamer is replaced by the connector complex.[15] DNA packaging is accompanied by the release of the scaffolding protein.[16] After the DNA incorporation the connector interacts with other tail components (gp11, gp12 and gp9) to secure the DNA (Figure 1 b) inside the head shell resulting in the mature virus (Figure 1 a3). This final, mature virus particle is then ready for further infection cycles by attaching to host cells (Figure 1 a4).[17] After attachment to the host, the entrance of the genome of phage ϕ 29 apparently shows a two step push–pull mechanism.[18] During the first stage of genome injection, the push stage carries about 65% of the DNA through the tail into the host. Afterwards, during the pull stage, a variety of proteins of the cytoplasm pull the remaining DNA inwards. Since the push stage would be triggered by releasing the internal pressure of the phage, it is essential to determine the pressurization, if any, of the viral particles.