The presynaptic filament is an obligatory intermediate in general genetic recombination. It is composed of a strand transferase protein polymerized along single-stranded DNA. In bacteriophage T4-infected cells, the presynaptic filament is composed of at least three proteins. In addition to the strand transferase (the uvsX protein), the uvsY (an accessory factor), and gene 32 (a helix-destabilizing factor), proteins also bind to the single-stranded DNA. In this report, we probe the assembly of the T4 presynaptic filament and the organization of the proteins in the complex. We find that interactions between the uvsY protein and the C terminus of the gene 32 protein are required to load UvsY onto gene 32 protein-covered DNA. Efficient binding of the uvsY protein to DNA is shown to be necessary for subsequent loading of the uvsX protein.