In recent years, a great deal of studies have focused on the possible toxicity of carbon nanotubes (CNT), as a result of their potential applications in the field of nanotechnologies. The investigation of spleen toxicity is part of the carbon nanotubes-induced toxicity assessment. In this study, we investigated the possible toxic effects of CNT on the rat spleen, after intraperitoneally (i.p.) administration of a single dose [1.5 ml; 2 mg multi-walled (MW) CNT per body weight (bw)] of multi-walled carbon nanotubes (exterior diameter 15–25 nm, interior diameter 10–15 nm, surface 88 m2 g–1) functionalized 1:1 with single-strand DNA (ss-DNA-MWCNT, 270 mg l–1). CNT functionalization with DNA determines a stable dispersion in the body fluids. For the detection of carbon nanotubes in the spleen, Raman spectroscopy, histopathologic examination, confocal microscopy and transmission electron microscopy (TEM) were performed at different time points (1, 6, 24, 48 and 144 h) after MWCNT administration. The dynamics of oxidative stress parameters (malondialdehyde, protein carbonyls and reduced glutathione), along with nitrosative stress parameters (nitric oxide, inducible NO synthase), the pro-inflammatory cytokines [interleukin-(IL)-1β] and the number of cells expressing caspase 3 and proliferating cell nuclear antigen (PCNA) were assessed. Our results indicate that, after i.p. administration, MWCNT translocate progressively in the spleen, with a peak of concentration after 48 h, and determine lymphoid hyperplasia and an increase in the number of cells which undergo apoptosis, in parallel with the enhancement of the mitosis in the white pulp and with transient alterations of oxidative stress and inflammation that need further investigations for a longer period of monitoring. Copyright © 2013 John Wiley & Sons, Ltd.