Interferon (IFN)-based therapy is the recommended treatment for hepatitis C virus. Because pegylated IFN (PEG-IFN) alfa-2a is administered subcutaneously, it is of interest to determine the proportion of the dose that is absorbed from the subcutaneous (SC) tissue and ultimately reaches systemic circulation. The goal of this study was to characterize the absolute bioavailability of PEG-IFN alfa-2a (40 kDa) after SC dosing (180 μg) and to evaluate the pharmacokinetics of PEG-IFN alfa-2a after intravenous (IV) and SC administration. In this parallel-group study, 18 participants were given a single IV dose of PEG-IFN alfa-2a 90 μg and 18 participants received PEG-IFN alfa-2a 180 μg SC. Serum concentrations of PEG-IFN alfa-2a were measured predose and serially until 312 hours after the first dose. Pharmacokinetic parameters (CL/F, volume of distribution, C max , and T max ) were estimated using noncompartmental methods. Bioavailability was calculated by using the following formula: (AUC SC /AUC IV ) · (dose IV /dose SC ). Eighteen healthy males received IV PEG-IFN alfa-2a, and an additional 18 healthy males received SC PEG-IFN alfa-2a. Subjects in each group had comparable mean weight, height, and body mass index. After IV administration of PEG-IFN alfa-2a (90 μg), there was a slow decline in serum concentration, the mean rate of systemic clearance was low at 126 mL/h, and the estimated mean volume of distribution at steady state was 9 L. After SC administration of PEG-IFN alfa-2a 180 μg, absorption was sustained, with mean T max occurring 102 hours after administration. The mean absolute bioavailability was 84%. A higher rate of influenza-like symptoms was observed after IV administration, along with decreased neutrophil counts, compared with subjects who underwent SC dosing. Approximately 84% of a SC-administered dose of PEG-IFN alfa-2a reached the systemic circulation in these male healthy volunteers. The slow absorption, restricted distribution, and slow elimination of PEG-IFN alfa-2a resulted in sustained serum levels throughout the 7-day dosing interval.