Interferon-α (IFN-α) is a key element in the defense against viral infection because, in addition to a direct antiviral effect, it exhibits potent immunostimulatory activity. To investigate the function of this cytokine in the woodchuck model of chronic hepatitis B, the woodchuck IFN-α gene (IFNA) family was cloned and examined. The data indicate that this is a multigenic family from which 12 IFNA functional sequences and four pseudogene sequences were isolated. The overall identity of the amino acid sequence among the members of the woodchuck IFN-α family is 85%, and the identity with the IFN-α family from other species such as mice and humans is 50%. The analysis of hepatic expression of IFNA genes showed that wIFNA5a was the subtype transcribed preferentially in the woodchuck liver. The wIFNA genes transcribed in the liver were tested in an eukaryotic expression system and were found to enhance 2–5-oligoadenylate synthetase (2–5-OAS) mRNA levels and to posses a potent antiviral activity. Cloning of woodchuck IFNA genes will allow testing diverse forms of IFN-α delivery as well as different combination therapies in woodchuck hepatitis virus infection, thus providing useful information for the design of new strategies for the treatment of patients with chronic hepatitis B. J. Med. Virol. 68:424–432, 2002. © 2002 Wiley-Liss, Inc.