We previously modulated, by conjugating a single cholesterol, plasma protein binding and liver cell uptake of a phosphorothioate oligodeoxynucleotide (PS-ODN). In this study, we investigated the biological fate of a PS-ODN, denoted ISIS-9389 (3',5'-bis-cholesteryl-conjugated ISIS 3082), provided with two cholesteryl moieties. After intravenous injection of into rats, [(3)H]ISIS-9389 was cleared from plasma with a half-life of 23.6 +/- 0.3 min. After 90 min (approximately 95% cleared), the liver contained 83.0 +/- 0.8% of the dose. Spleen and bone (marrow), which constitute with the liver the reticuloendothelial system, contained 3.1 +/- 0.3 and 4.3 +/- 0.2%, respectively. All other tissues accumulated together <5% of the dose. The hepatic uptake of [(3)H]ISIS-9389 occurred mainly by endothelial cells (51.9 +/- 6.4% of the liver uptake). Parenchymal and Kupffer cells were responsible for 24.9 +/- 7.7 and 23.3 +/- 2.5%, respectively. Preinjected polyinosinic acid and polyadenylic acid reduced hepatic uptake, albeit the latter was less effective. This finding suggests implication of (multiple) scavenger receptors in liver uptake of ISIS-9389. The interaction of ISIS-9389 with plasma proteins, analyzed by size exclusion chromatography, differs from that of unconjugated PS-ODN and PS-ODN with a single cholesterol. Plasma-incubated ISIS-9389 was mainly recovered as a high molecular weight complex. In conclusion, conjugation of PS-ODNs with two cholesteryl moieties results in almost quantitative uptake by the liver. The liver targeting exceeds the already impressive gain in liver uptake achieved by conjugation of a single cholesterol, and is expected to increase the therapeutic activity against liver-associated targets and reduce side effects in nonhepatic tissues.