Purpose: To investigate the influence of chromatin organization and scavenging capacity in relation to irradiation temperature on the induction of double-strand breaks (DSB) in structures derived from human diploid fibroblasts. Materials and methods: Agarose plugs with different chromatin structures (intact cells±wortmannin, permeabilized cells with condensed chromatin, nucleoids and DNA) were prepared and irradiated with X-rays at 2 or 37°C and lysed using two different lysis protocols (new ice-cold lysis or standard lysis at 37°C). Induction of DSB was determined by constant-field gel electrophoresis. Results: The dose-modifying factor (DMFtemp) for irradiation at 37 compared with 2°C was 0.92 in intact cells (i.e. more DSB induced at 2°C), but gradually increased to 1.5 in permeabilized cells, 2.2 in nucleoids and 2.6 in naked DNA, suggesting a role of chromatin organization for temperature modulation of DNA damage. In addition, DMFtemp was influenced by the presence of 0.1 M DMSO or 30 mM glutathione, but not by post-irradiation temperature. Conclusion: The protective effect of low temperature was correlated to the indirect effects of ionizing radiation and was not dependent on post-irradiation temperature. Reasons for a dose modifying factor <1 in intact cells are discussed.