Intraspecies transfection of ribosomal RNA genes (rDNA) into human fibroblasts was used to reexamine characteristics of the rDNA complex which lead to participation in nucleolar formation. Two constructs of human rDNA were analyzed. One included an intact transcribed spacer and promoter region and most of the nontranscribed spacer (NTS) region. The second contained the transcribed spacer and much of the NTS region, but lacked the transcription initiation region. Transfected rDNA, shown to be present in single or low copy number and containing the promoter region, was present in nucleoli. In contrast, transfected rDNA lacking the promoter did not participate in nucleolar organizing region (NOR) formation. We conclude from this study that tandem repetition, a feature of rDNA organization, is not an absolute requirement for rDNA function and that the presence of the rDNA transcription initiation region and promoter is necessary in establishing an active NOR.