Helper-dependent (HD) adenoviral (Ad) vectors, in which all viral coding sequences are deleted, have been generated. We show here that intravenous delivery of a mouse EPO (mEPO) expression cassette cloned in an HD vector in immunocompetent mice is effective and long lasting, but not permanent. A precise dose-response relationship between the dose of injected virus and stable EPO serum levels was observed, together with a 100-fold increase in gene expression per infectious particle when compared with a first-generation Ad vector bearing the same cassette. As a direct consequence, therapeutic increases in hematocrit that lasted more than 6 months were achieved with minute amounts of virus, which caused no detectable production of neutralizing antibodies. Intravenous readministration of the HD-mEPO vector in the same mice was as effective as in naive animals without any need for prior immunosuppression. Finally, HD-mEPO injection in subtotally nephrectomized rats improved the anemic status induced by surgery. HD Ad vectors are thus excellent tools for EPO gene therapy.