BACKGROUND We have previously described a five-plasmid HIV-1 vector system that utilises a codon-optimised gagpol gene. While this system was shown to be safer than systems using proviral type helpers, the titre of virus produced was relatively low. Therefore, a process of optimising all aspects of virus production was initiated. METHODS A systematic approach was taken to the optimisation of virus production by transient expression using a five-plasmid packaging system. Codon-manipulation was used to reduce homology between helper and vector constructs. Ultrafiltration and ultracentrifugation were used for large-scale virus production. RESULTS We describe codon-optimised reading frames for Tat and Rev and the optimisation of virus production. The optimisation process resulted in an increase in virus titre of 7- to 8-fold. Several other approaches to increasing viral titre described by others proved ineffective in our system after it had been optimised. In addition, we show that by varying the ratio of the GagPol helper construct to vector, the infectivity of the virus could be controlled. The use of a novel codon-optimised HIV-1 GagPol expression construct with reduced homology to vector sequences significantly reduced transfer of gagpol sequences to transduced cells. Virus could be collected in serum-free medium without a significant loss of titre, which facilitated subsequent processing. Processing using a combination of ultrafiltration and ultracentrifugation allowed efficient and rapid processing of litre volumes of virus supernatant. CONCLUSIONS By taking a systematic approach to optimising all aspects of our five-plasmid lentiviral vector system we improved titre, safety, large-scale production, and demonstrated that infectivity could be specifically controlled.