This study examined the plasma pharmacokinetics, tissue distribution, and metabolism of three second generation antisense oligonucleotides in monkeys. Three groups of monkeys were treated with 10 mg/kg of each test compound by a single 2‐h intravenous infusion. Oligonucleotide concentrations were measured in plasma, tissues, and urine using capillary gel electrophoresis (CGE). HPLC‐MS was used to identify the metabolite(s) of the study compounds. Plasma–concentration–time profiles after infusion for the two phosphorothioate oligonucleotides were mono‐exponential, but was bi‐ exponential for the phosphodiester oligonucleotide. Plasma clearance for the phosphodiester oligonucleotide was four‐ to sevenfold higher than the two phosphorothioate oligonucleotides, which was attributed to the plasma protein binding and reduced nuclease resistance. 2′‐O‐(2‐methoxy) ethyl (MOE) modification at both 3′ and 5′ ends of a phosphorothioate oligonucleotide greatly enhanced the resistance to nucleases in plasma and tissue. MOE modification only at the 3′ end enhanced the resistance to nucleases in plasma, but only moderately enhanced the resistance to nucleases in tissues. Urinary excretion was a minor elimination pathway for the phosphorothioate oligonucleotide, but was a major elimination pathway for the phosphodiester oligonucleotide. The results characterize the relationships between structure and disposition and will direct future modifications for therapeutic use. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:48–59, 2004