The reverse genetics system for influenza A viruses described by Hoffmann et al. (Virology 267(2):310-317, 2000, Proc Natl Acad Sci USA 97(11):6108-6113, 2000, ArchVirol 146(12):2275-2289, 2001) is one of the most commonly used. However, this cloning strategy is rather time-consuming and lacks a selection marker to identify positive clones carrying viral genes. We report here the optimization of the cloning protocol of viral genes into pHW2000 by (i) introducing a selection marker and (ii) simplifying the cloning strategy: now the cloning reaction takes only a few minutes and, in addition, is independent of internal restriction sites for BsmBI/Esp3I, BsaI or AarI. In order to accelerate the whole cloning protocol for the generation of recombinant viruses, we first introduced a lacP/Z-element (lac-promoter/lacZα-fragment) between the two BsmBI sites of pHW2000 to allow selection of positive clones by blue/white screening. Then we optimized the digestion/ligation-protocol: In our system, enzymatic digestion and ligation of PCR products into the vector is performed in a single "one-tube" reaction. Due to this strategy, time and material consumption is reduced by a great amount, as vector and cDNA do not have to be digested and purified prior to the ligation. Therefore, this one-tube reaction yields positive clones with high efficiency and fidelity, again saving time and material, which were formerly required for screening and analyzing clones. Finally, to add more versatility to the system, we also created an entry vector based on TA-cloning. This entry vector provides several advantages: inserted genes can easily be modified, e.g., by site-directed mutagenesis or tag attachment, and then subcloned into pHW2000 or other plasmids containing a similar cloning site (e.g., our modified pCAGGS-Esp-blue) by the same rapid and reliable one-tube reaction protocol described here. In fact, the presented protocol is suitable to be adapted to other reverse genetics systems (e.g., those for members of the order Mononegavirales or the family Bunyaviridae) or cloning of genes in general.