To investigate the pharmacokinetics, safety, and tolerability of GS-9851 (formerly PSI-7851), a new nucleotide analog inhibitor of hepatitis C virus (HCV), we conducted a double-blind, parallel, placebo-controlled, randomized, single-ascending-dose study. Healthy subjects received oral doses of 25 to 800 mg GS-9851. Peak concentrations of GS-9851 in plasma were achieved more rapidly than those of the metabolites GS-566500 (formerly PSI-352707) and GS-331007 (formerly PSI-6206), with time to maximum concentration of drug in plasma (t(max)) values of 1.0 to 1.8 h, 1.5 to 3.0 h, and 3.0 to 6.0 h, respectively. The majority of systemic drug exposure was from the nucleoside GS-331007, with maximum concentration of drug in plasma (C(max)) and area under the concentration-time curve to the last measurable concentration (AUC(0-t)) values at least 7- and 41-fold higher, respectively, than those obtained for GS-9851 after adjusting for differences in molecular weight. The terminal elimination half-life (t(1/2)) of GS-331007 increased with the dose, achieving a t(1/2) of 25.7 h at 800 mg GS-9851. Dose proportionality was not observed for GS-331007. The majority of drug recovered in urine was in the form of GS-331007, with the percentage of this metabolite in urine samples ranging from 57% to 27% with increasing dose. GS-9851 was generally well tolerated, with no maximum tolerated dose identified. In conclusion, GS-9851 and its metabolites demonstrated a favorable pharmacokinetic profile consistent with once-daily dosing, and therefore, further clinical studies evaluating GS-9851 in HCV-infected patients are warranted.