Administration of interferon (IFN) 3 times weekly in patients with chronic hepatitis C (CHC) is associated with low sustained responses, which may be, in part, related to this regimen's inability to maintain IFN concentrations sufficient to suppress viral replication. An enhanced IFN molecule produced by the covalent attachment of a branched 40-kd polyethylene glycol moiety to IFN α-2a (PEG[40kd] IFN α-2a) exhibits sustained absorption, a restricted volume of distribution, and reduced clearance compared with unmodified IFN α-2a. One hundred fifty-nine patients with CHC participated in a randomized, ascending-dose (45 or 90, 180, 270 μg) study comparing PEG(40kd) IFN α-2a administered once weekly with 3 MIU IFN α-2a administered 3 times weekly for 48 weeks to determine the most appropriate PEG(40kd) IFN α-2a dose for subsequent clinical trials. Efficacy was assessed by measuring hepatitis C virus (HCV) RNA following a 24-week treatment-free period. Sustained virological responses for PEG(40kd) IFN α-2a once weekly were 10% (45 μg; not significant), 30% (90 μg; P = .009), 36% (180 μg; P = .0006), and 29% (270 μg; P = .004), compared with 3% for the 3-times-weekly 3-MIU IFN α-2a regimen. The types and frequencies of adverse events and laboratory abnormalities were similar among all groups. In conclusion, once-weekly PEG(40kd) IFN α-2a was associated with a higher number of sustained virological responses compared with IFN α-2a 3 times weekly in patients with CHC, but had a similar safety profile. The 180-μg PEG(40kd) IFN α-2a dose appeared to be the optimal dose based on sustained virological response and its associated side-effect profile.