Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression. Currently, the most widely used system for HD vector production avoids significant contamination with helper virus by using producer cells stably expressing a nuclear-targeted Cre recombinase and an engineered first-generation helper virus with parallel loxP sites flanking its packaging signal. The system requires a final, density-based separation of HD and residual helper viruses by ultracentrifugation to reduce contaminating helper virus to low levels. This separation step hinders large-scale production of clinical-grade HD virus. By using a very efficient recombinase, in vitro-evolved FLPe (ref. 14), to excise the helper virus packaging signal in the producer cells, we have developed a scalable HD vector production method. FLP has previously been shown to mediate maximum levels of excision close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD plasmid backbone, the FLPe-based system reproducibly yielded HD virus with the same low levels of helper virus contamination before any density-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography-based virus purification.