Investigation of the mechanisms underlying the biological effects induced by densely ionizing radiation has relevant implications in both radiation protection and therapy. In particular, the possible advantages of hadrontherapy with respect to conventional radiotherapy in terms of high conformal tumor treatment and sparing of healthy tissues are well known. Further improvements are limited by lack of radiobiological knowledge, particularly about the specific cellular response to the damage induced by particles of potential interest for tumor treatment. This study compares early and late effects induced in AG01522 normal human primary fibroblasts by γ-rays and C-ions having E ∼ 45 MeV/u at the cell entrance, corresponding to LET (in water) ∼ 49 keV/μm. Different end points have been investigated, namely: cell killing and lethal mutation, evaluated as early and delayed reproductive cell death, respectively; chromosome damage, as measured by micronuclei induction (MN); DNA damage, in terms of DSB induction and repair, as measured by the H2AX phosphorylation/dephosphorylation kinetics. Linear dose-response relationships were found for cell killing and induction of lethal mutations, with RBEs of about 1.3 and 1.6 respectively, indicating that the presence of genomic instability is greater in the progeny of C-ions irradiated cells. H2AX phosphorylation/dephosphorylation kinetics have shown a maximum foci number at 30 min after irradiation, higher for γ-rays than for C-ions. However, in the first 12 h the fraction of residual γ-H2AX foci was higher for C-ions irradiated cells, indicating a lower removal rate, possibly related to multiple/more complex damage along the particle track, with respect to the sparse lesions produced by γ-rays. MN induction, observed after 72 h from irradiation, was also greater for C-ions. Overall, these data indicate a more severe DNA damage induced by 45 MeV/u C-ions with respect to γ-rays, likely responsible of an increased cellular misrepair, leading to the greater observed levels of chromosome damage and, eventually, of genomic instability. They give strong support to the idea that higher damage severity at molecular level, determined by the typical deposition pattern of densely ionizing radiation, is the earliest relevant factor for the more severe late effects at cellular level. © 2013 AIP Publishing LLC.