We have previously demonstrated that the immune response to an unmethylated cytidine-guanosine (CpG)-containing oligonucleotide (ODN) is greatly enhanced when encapsulated in a lipid nanoparticle (LN-CpG ODN). In this study, the pharmacokinetics, biodistribution and cellular uptake of LN-CpG ODN following intravenous (i.v.) and subcutaneous (s.c.) administration was characterized and correlated with immunostimulatory activity. It is shown that, despite dramatic differences in tissue distribution profiles and considerable differences in uptake by CD11c-positive, CD11b-positive, Mac-3-positive and CD45R/B220-positive cells following i.v. and s.c. administration, the resultant immune response is very similar with respect to levels of cellular activation (DX5, Mac-3, CD11b, CD45/B220, CD4, CD8 and CD11c) and cytolytic activity of immune cells [natural killer (NK) cells and monocytes/macrophages] in the spleen and blood compartments. Some differences in response kinetics and antibody-dependent cellular cytotoxicity (ADCC) activity were noted in the peripheral blood NK cell population. Analyses of particle biodistribution and cell types involved in uptake leads to the conclusion that the inherent ability of antigen-presenting cells (APCs) to sequester LN-CpG ODN results in efficient uptake of the particle, even when present at very low concentrations, leading to similar responses following i.v. and s.c. administration. These results contrast with the behavior of free CpG ODN, for which distinctly different immune responses are observed following i.v. or s.c. administration.