A sensitive system for the assay of bacteriophage phi 29 assembly in vitro was developed using 12 recombinant proteins and synthetic pRNA. This system detected in vitro assembled infectious phages up to 10(7) plaque forming units (PFU) per milliliter without any background. phi 29 DNA-gp3 concentration dependence in phage assembly was found to be first order, while the DNA-packaging protein gp16 dependence was higher order. The requirement for specific phi 29 pRNA for phi 29 DNA packaging was confirmed by the finding that no plaques were formed when only Escherichia coli RNAs were present. The activity of a mutant pRNA, with 10(5)-fold reduction in DNA packaging efficiency, was also demonstrated. Additionally, the tail proteins were found to have dual roles, one acting as phage tails and the other stabilizing the DNA-gp3 filled capsids.