Rhodobacter capsulatus produces a gene transfer agent (GTA) called RcGTA. RcGTA is a phage-like particle that packages R. capsulatus DNA and transfers it to other R. capsulatus cells. We quantified the relative frequency of packaging for each gene in the genome by hybridization of DNA from RcGTA particles to an R. capsulatus microarray. All genes were found within the RcGTA particles. However, the genes encoding the RcGTA particle were under-packaged compared with other regions. Gene transfer bioassays confirmed that the transfer of genes within the RcGTA structural cluster is reduced relative to those of other genes. Single-cell expression analysis, by flow cytometry analysis of cells containing RcGTA-reporter gene fusion constructs, demonstrated that RcGTA gene expression is not uniform within a culture. This phenomenon was accentuated when the constructs were placed in a strain lacking a putative lysis gene involved in RcGTA release; a small subpopulation was found to be responsible for ∼ 95% of RcGTA activity. We propose a mechanism whereby high levels of RcGTA gene transcription in the most active RcGTA-producing cells cause a reduction in their packaging frequency. This subpopulation's role in producing and releasing the RcGTA particles explains the lack of observed cell lysis in cultures.