Recombinant vaccinia viruses (VVs) are widely used as expression vectors in molecular biology and immunology and are now under evaluation for gene therapy. The current techniques for inserting foreign DNA into the large VV genome are based on either homologous recombination between transfer plasmids and VVgenomes or direct DNA ligation and packaging using replication-deficient poxviruses. Here, we describe efficient new versions of both methods that produce 90%-100% of the recombinant viruses. In the new homologous recombination method, VV DNA "arms" obtained by NotI digestion and intact transfer plasmids were used for co-transfection. In the direct DNA ligation method, foreign DNA was inserted into a unique NotI restriction site of the VVgenome. In both methods, the generation of recombinant viruses was carried out in cells infected with a non-replicating, psoralen-UV (PUV)-inactivated helper VV. The convenience of these new techniques is demonstrated by the construction of recombinant VVs that produce E. coli beta-galactosidase. An important feature of these strategies is that any VV strain can be used as a helper virus after PUV inactivation.