It has recently been shown that hepatitis B virus (HBV)-based vectors are suitable for a hepatocyte specific gene transfer. As candidate vectors for gene therapy, however, they should no longer express any viral gene products. In addition, the insertion of promoters that do not originate from HBV is needed to allow a variation of the level of gene expression. Furthermore, production of high-titer stocks is required. To eliminate viral gene expression, we knocked out all HBV open reading frames (ORFs) in the transfer construct used to express recombinant HBV RNA. To minimize the chance of homologous recombination, we generated an improved helper construct. With these constructs, recombinant viruses containing single or combined knockouts of the viral ORFs were produced at titers equal to wild-type HBV produced in parallel. We identified a site in the HBV genome that allows insertion of foreign promoter elements without interfering with maturation of infectious HBV particles. Successful gene transfer was demonstrated on infection of primary human hepatocytes using recombinant HBV in which all viral ORFs were knocked out and a foreign promoter controlled transgene expression. These improvements represent a major step toward the development of HBV vectors as candidates for human gene therapy.