Cytoplasmic extracts from cultured mouse cells contain the factor(s) required for specific transcription initiation of rDNA by RNA polymerase I. Prior to transcription the essential proteins bind to the ribosomal gene and remain bound to the template for several rounds of transcription. The assembly of these preinitiation complexes in vitro has been demonstrated by kinetic analysis of the transcription reaction and by competition experiments. Complex formation involves an initial, rapid binding of transcription factor(s) to rDNA sequences followed by additional events which arrange the DNA-protein complex into a transcriptionally active state. Once the complexes have formed they persist for at least 2 hours in vitro and are resistant to elevated salt concentrations. The assembly of the complexes was inhibited when the template DNA was incubated with histones prior to the addition of S-100 extract. If, however, preinitiation complex formation was allowed to occur before the addition of histones, the interference of histones with specific transcription was much less pronounced.