We report here our initial success in using fluorescence energy transfer to map the position of the subunits of the DNA polymerase III holoenzyme within initiation complexes formed on primed DNA. Using primers containing a fluorescent derivative 3 nucleotides from the 3'-terminus and acceptors of fluorescence energy transfer located on Cys333 of the beta subunit, a donor-acceptor distance of 65 A was measured. Coupling this distance with other information enabled us to propose a model for the positioning of beta within initiation complexes. Examination of the fluorescence properties of a labeled primer with the unlabeled beta subunit and other assemblies of DNA polymerase III holoenzyme subunits allowed us to distinguish all of the known intermediates of the holoenzyme-catalyzed reaction. Specific fluorescence changes could be assigned for primer annealing, Escherichia coli single-stranded DNA-binding protein binding, 3'----5' exonucleolytic hydrolysis of the primer, DNA polymerase III* binding, initiation complex formation upon the addition of beta in the presence of ATP, and DNA elongation. These fluorescence changes are sufficiently large to support future detailed kinetic studies. Particularly interesting was the difference in fluorescence changes accompanying initiation complex formation as compared to binding of DNA polymerase III holoenzyme subunit assemblies. Initiation complex formation resulted in a strong fluorescence enhancement. Binding of DNA polymerase III* led to a fluorescence quenching, and transfer of beta to primed DNA by the gamma delta complex did not change the fluorescence. This demonstrates a rearrangement of subunits accompanying initiation complex formation. Monitoring fluorescence changes with labeled beta, we have determined that beta binds with a stoichiometry of one monomer/primer terminus.