An in situ single-pass perfusion model was used to assess the effect of chemical modification and length on permeability and absorption of various oligonucleotides in rat intestine. Phosphorothioate oligodeoxynucleotides (PS-ODN) were compared with oligoribonucleotides with 2'-methoxyethyl (MOE) or 2'-O-methyl (OMe) modifications. A 25-mer PS-OMe-modified oligonucleotide showed relatively poor permeability in this model, as did unmodified 20-mer PS-ODN (permeability coefficient [P(eff)] = 2-8 X 10(-6)cm/sec). Modifying some or all of the oligonucleotides with 2'-MOE groups on deoxyribose and 5'-methylation of the cytosines substantially increased intestinal permeability of oligonucleotides. Both partially and fully modified PS-MOE oligonucleotides showed a (2-4)-fold increase in permeability as compared with unmodified PS-ODN. The presence of a phosphodiester backbone in MOE-modified compounds led to further increases in intestinal permeability. PS-MOE composed of 6, 8, 10, 12, 14, 16, 18, 20, and 22 nucleotides were also examined. It was found that the permeability of these oligonucleotides increased linearly with decreasing length.